Polynucleotides encoding apoa1-pon1 fusion polypeptides

ABSTRACT

Compositions and methods relating to ApoA-1 fusion polypeptides are disclosed. The fusion polypeptides include a first polypeptide segment corresponding to an ApoA-1 polypeptide or ApoA-1 mimetic, and may also include a dimerizing domain such as, e.g., an Fc region, which is typically linked carboxyl-terminal to the first polypeptide segment via a flexible linker. In some embodiments, the fusion polypeptide further includes a second polypeptide segment located carboxyl-terminal to the first polypeptide segment and which confers a second biological activity (e.g., an RNase, paraoxonase, platelet-activating factor acetylhydrolase, cholesterol ester transfer protein, lecithin-cholesterol acyltransferase, polypeptide that specifically binds to proprotein convertase subtilisin/kexin type 9, or polypeptide that specifically binds to amyloid beta). Also disclosed are dimeric proteins comprising first and second ApoA-1 fusion polypeptides as disclosed herein. The fusion polypeptides and dimeric proteins are useful in methods for therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/909,314, filed Mar. 1, 2018, which is a continuation-in-part ofInternational Application No. PCT/US2016/050405, filed Sep. 6, 2016,which claims the benefit of U.S. Provisional Application No. 62/215,256,filed Sep. 8, 2015. Each of the foregoing applications is incorporatedby reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Nov. 26, 2021, isnamed “TRP_0130 US_20211126_Seq_Listing_ST25” and is 177,536 bytes insize.

BACKGROUND OF THE INVENTION Apolipoprotein A-I (ApoA-1) and High DensityLipoprotein (HDL)

Cardiovascular disease is the leading cause of mortality in manynations, accounting for approximately 16.7 million deaths each yearworld-wide. The most common consequences of cardiovascular disease aremyocardial infarction and stroke, which have a common underlyingetiology of atherosclerosis.

Epidemiological studies since the 1970's have shown that low levels ofhigh density lipoprotein (HDL) is associated with increased risk formyocardial infarction. This has led to multiple approaches to newtherapies targeting HDL (see Kingwell et al., Nature Reviews DrugDiscovery 13:445-64, 2014) and to a consensus view that the process ofreverse cholesterol transport (RCT) is central to beneficial HDLactivity rather than simply an increase in HDL without RCT. For example,in clinical trials so far, drugs that increase HDL by inhibition of RCTwith inhibitors of cholesterol ester transfer protein (CETP) have notbeen efficacious. Further, it has more recently been realized thatmeasuring levels of HDL is not sufficient to determine its function inpatients because HDL is damaged by oxidation and glycation, includingduring chemotherapy and in patients with neurodegenerative disorders.See Keeney et al., Proteomics Clin Appl. 7:109-122, 2013.

Apolipoprotein A-1 (ApoA-1) is the principal protein component of HDL.Phillips, Journal of Lipid Research 54:2034-2048, 2013. Human ApoA-1 isa 243 amino acid protein, with a series of eight 22-mer and two 11-meramphipathic α-helices spanning residues 44-243. Lund-Katz and Phillips,Subcell Biochem. 51:183-227, 2010. The helices in the amino-terminaltwo-thirds of the molecule form a helix bundle structure, whereas thecarboxyl-terminal region forms a separate, relatively disorganizeddomain important for lipid binding. The interaction of the C-terminalsegment with lipids induces conformational changes in the ApoA-1structure, increasing the α-helix content of the molecule and allowingsubsequent opening of the N-terminal helix bundle. See id. The lipidaffinity of ApoA-1 confers detergent-like properties, and it cansolubilize phospholipids to form discoidal HDL particles containing asegment of phospholipid bilayer and two ApoA-1 molecules arranged in ananti-parallel, double-belt conformation around the edge of the disc.Phillips, supra. The conformational adaptability ApoA-1 also confersstability to HDL particles, including discoidal particles of differentsizes as well as spherical HDL particles. See id. These characteristicsallow ApoA-1 to partner with ABCA1 in mediating efflux of cellularphospholipid and cholesterol and the production of stable HDL particles.See Phillips, supra; Lund-Katz and Phillips, supra.

Due to its important role in HDL particle formation and function, ApoA-1has become the focus for several HDL-targeted therapeutic strategies.Drugs including niacin and fibrates that increase synthesis of ApoA-1,however, also decrease the concentration of very low densitylipoproteins (VLDL) and are thus not specific for HDL. Clinical trialsof niacin were halted due to lack of efficacy, whereas fibrates thatactivate peroxisome proliferator activated receptors (PPARs) were foundto cause a 10% reduction in major cardiovascular events (p<0.05) and a13% reduction in coronary events (p<0.0001) in a meta-analysis. See Junet al., Lancet 375:1875, 2010. Because more effective therapies areneeded, there are several other orally active drugs that increase ApoA-1in preclinical development. See Kingwell et al., supra.

An alternative approach to increasing ApoA-1 is by direct injection ofthe purified protein. See, e.g., Kingwell et al., Circulation 128:1112,2013. ApoA-1 has been purified from human plasma (reconstituted HDL) andtested in clinical trials. Recombinant ApoA-1 has also been expressed inboth bacterial and mammalian expression systems and tested in clinicaltrials. These studies have shown that infusion of ApoA-1, reconstitutedwith phospholipids into pre-β HDL, causes reduction of plaque volume andimprovement in plaque morphology as measured by intravascular ultrasound(IVUS) after small (47-60 patients) clinical trials. While promising,use of natural or recombinant ApoA-1 has several limitations, includinga requirement for weekly administration due to a short ApoA-1 half-lifeand a high cost of manufacturing.

Recombinant ApoA-1 Milano, a highly active ApoA-1 mutant, was expressedin bacterial cells and tested in clinical trials in patients with acutecoronary syndromes (see Nissen et al., JAMA 290:2292, 2003), wherereduction in plaque volume was seen. While this study is considered thefirst to directly test and confirm the HDL hypothesis, ApoA-1 producedin bacterial systems has not progressed due to low expression levels andhigh manufacturing costs. Recombinant ApoA-1 produced in mammalian cellshas progressed further in clinical trials, including recently completedphase II studies. CER-001, in development by Cerenis Therapeutics, is arecombinant ApoA-1 produced by mammalian cells and formulated withspecific lipids to form pre-β-like HDL particles. According to Cerenis,CER-001 met its primary end point of a reduction in carotid plaquevolume measured by MRI in patients with familial hypercholesterolaemiain the MODE trial (NCT01412034). In the CHI-SQUARE trial (NCT01201837),Cerenis announced that CER-001 reduced plaque volume versus baseline inpatients with acute coronary syndrome, but the reduction was notsignificant versus placebo.

In another study in macaques, ApoA-1 Milano, reconstituted with lipids(POPC), was infused at relatively high doses (30, 100, and 300 mg/kg)given every second day for 21 infusions. Kempen et al., J. Lipid. Res.54:2341-2353, 2013. Drug infusion quickly decreased the endogenouscholesterol esterification rate, increased the formation of largeApoE-rich particles due to lack of LCAT activation, and caused a largeincrease in free cholesterol due to sustained stimulation ofABCA1-mediated efflux. See id. These results show that infusion of largeamounts of reconstituted ApoA-1 Milano disrupt HDL metabolism byenhancing cholesterol efflux without the ability to process it throughthe normal metabolic pathways.

While the prospects for HDL infusion therapy are very promising, thereis a need for improved recombinant ApoA-1 molecules that overcome someof the limitations of current approaches. Several recombinant ApoA-1fusion proteins have been produced, including ApoA-1 produced inbacteria with a His tag to simplify purification. See, e.g., Prieto etal., Protein J. 31:681-688, 2012; Ryan et al., Protein Expr. Purif.27:98-103, 2003. In another example, IFNα was attached to the aminoterminus of ApoA-1 through a 3aa (Gly Ala Pro) linker. See Fioravanti etal., J. Immunol. 188:3988-3992, 2012. The linker in this construct wascreated by the choice of restriction enzymes, and the fusion protein wastested by adenovirus delivery to target to the liver and reduce thetoxicity of IFNα therapy. ApoA-1 has also been fused with an Fc domain(ApoA-1-Ig) and is available commercially from Creative Biomart (cat.No. APOA-1-33H) and from Life Technologies (Cat #10686-HO2H-5). However,this ApoA-1-Ig molecule has very low functional activity (see Example1).

Additional recombinant ApoA-1 fusion proteins include anti-CD20scFv-ApoA-1 (Crosby et al., Biochem. Cell Biol. 10:1139/bcb, 2015),IL-15-ApoA-1 (Ochoa et al., Cancer Res. 73:139-149, 2013), and atrimeric ApoA-1 fusion protein made by the addition of the trimerizationdomain of human tetranectin (Graversen et al., J. CardiovascularPharmacol. 51:170-77, 2008). In these examples, the fusion was at theN-terminus of ApoA-1.

The trimeric tetranectin-ApoA-1 (TN-ApoA-1) was effective in reversecholesterol efflux and its half-life in mice was increased to 12 hoursversus three hours for monomeric ApoA-1. See Gaversen et al., supra. Inan aggressive model of atherosclerosis (LDLR−/− mice fed a high-fatdiet), trimeric TN-ApoA-1 slowed progression of lesions in the aorticroots. See id. Recent studies in nonhuman primates, however, showed thatmultiple infusions of lipidated TN-ApoA-1 were not well tolerated andresulted in high immunogenicity and lipid accumulation. SeeRegeness-Lechner et al., Toxilogical Sciences 150:378-89, 2016. Thetrimer fusion protein was complexed with phospholipids and injected atconcentrations of 100 mg/kg and 400 mg/kg every four days for threeweeks, followed by a six week recovery period. After multiple infusionsof lipidated TN-ApoA-1, clinical condition deteriorated and wasaccompanied by changes indicative of a progressive inflammatoryresponse, increased levels of cytokines, C-reactive protein andvascular/perivascular infiltrates in multiple tissues. Rapid formationof antidrug antibodies occurred in all animals receiving lipidatedTN-ApoA-1. See id. The accumulation of trimeric TN-ApoA-1 in tissues ofthe treated animals resembles fibril formation and deposition of ApoA-1in patients who have mutations near the N-terminus. See Mizuguchi etal., J. Biol. Chem. 290:20947-20959, 2015; Das et al., J. Mol. Biol.2015.10.029; Obici et al., Amyloid 13:191-205, 2006.

Current forms of ApoA-1 in clinical development require formulation withspecific lipids into preβ-like HDL particles prior to infusion, becausethe half-life of ApoA-1 in the absence of lipids is very short. SeeNanjee et al, Arterioscler Thromb Vasc Biol 16:1203-1214, 1996 (showingthat lipid-free ApoA-1 has a half-life of only 2-2.3 hours after eitherbolus or slow infusion in humans). After lipid formulation, half-lifeincreases to about 48 hours, so frequent (weekly) administration isstill required.

ApoA-1 therapy has also shown significant benefit in improving insulinsensitivity and glucose uptake (see Drew et al., Nature ReviewsEndocrinology 8:237, 2012), and may be useful in patients with diabetesand with NASH (non-alcoholic steatohepatitis). In addition, ApoA-1 bindsamyloid-beta and prevents neurotoxicity in cultured hippocampal neuronalcells. See Koldamova et al., Biochemistry 40:3553, 2001; Paula-Lima etal., Int. J. Biochem. Cell Biol. 41:1361, 2009. Further, ApoA-1polymorphisms are linked to risk for Alzheimer's disease and ApoA-1 isfound at decreased levels in patients with neurodegenerative disorders.See Keeney et al., Proteomics Clin. Appl. 7: 109-122, 2013).

Efficacy of ApoA-1 therapy has also been demonstrated in animal modelsof cancer. One study examined the effect of ApoA-1 infusion on growth oftumors in mice. See Zamanian-Daryoush et al., J. Biol. Chem.288:21237-21252, 2013. Zamanian-Daryoush et al. found that ApoA-1potently suppresses tumor growth and metastasis in multiple syngeneictumor models, including B16F10L malignant melanoma and Lewis Lungcarcinoma. The effect of ApoA-1 was due to modulation of the immuneresponse. Recruitment and expansion of myeloid-derived suppressor cells(MDSC) in the tumors was inhibited. There was also inhibition of tumorangiogenesis and the matrix-degrading protease MMP-9. In contrast,ApoA-1 therapy increased CD11b macrophages and increased amounts ofIFNγ, IL-12b, and CXCL10, markers of a Th1 response supporting T cellactivation. The authors showed that T cells were required for the potentsuppressive effect of ApoA-1 on tumor growth, and ApoA-1 therapy causeda specific increase in CD8⁺ T cells in the tumors. See id. While theresults of Zamanian-Daryoush et al. are promising, the study used highdoses of lipid-free ApoA-1 to achieve the observed effects (15 mg everysecond day per mouse), see id., which was likely required because of theshort half-life of ApoA-1.

Another cancer study showed that ApoA-1 and mimetic peptides (L-4F,D-4F, L-5F) inhibit tumor development in a murine model of ovariancarcinoma. See Su et al., Proc. Natl. Acad. Sci. USA 107:19997-20002,2010. Su et al. found that ApoA-1 overexpression in transgenic mice, orpeptide mimetic administration, reduced stimulatory phospholipids,implicating an additional mechanism for inhibition of tumor growth. Seeid.

Studies have also suggested a role for ApoA-1 in the pathogenesis ofmultiple sclerosis (MS). In particular, ApoA-1 expression was shown tobe lower in MS patients compared to healthy controls, and primaryprogressive MS patients had less plasma ApoA-1 than patients with otherforms of MS. See Meyers et al., J. Neuroimmunol. 277:176-185, 2014.Using experimental allergic encephalomyelitis (EAE) as a model for MS,mice deficient in ApoA-1 exhibited worse clinical disease and moreneurodegeneration compared to wild-type animals. The authors suggestthat agents that increase ApoA-1 levels are possible therapies for MS.See id. Another MS study found that the ApoA-1 promoter polymorphismA-allele, associated with elevated ApoA-1 levels, is correlated withimproved cognitive performance in patients with MS; A-allele carriersdisplayed overall superior cognitive performance and had a three-folddecreased overall risk of cognitive impairment. See Koutsis et al.,Mult. Scler. 15:174-179, 2009.

Peptide Mimetics

ApoA-1 mimetic peptides have shown efficacy in a number of animal modelsof disease and have properties that make them attractive as potentialtherapeutic agents. See, e.g., Reddy et al., Curr. Opin. Lipidol.25:304-308, 2014 and White et al., J. Lipid. Res. 55:2007-2021, 2014.Peptide 4F has been tested in high risk patients with coronary arterydisease. Several ApoA-1 mimetic peptides that are resistant to oxidationhave been described in the past several years. While these α-helicalpeptides show activity in animal models, they require daily dosingbecause of their short half-life. In addition, toxicity, includingmuscle toxicity and hypertriglyceridemia, have been seen inpeptide-treated animals (these toxicities have been seen in mice treatedwith ApoA-1). Advances to reduce toxicity by sequence design and toreduce cost of peptide production were described. See, e.g., Bielicki,Curr. Opin. Lipidol. 27:40-46, 2016. Another approach has been tosynthesize D-peptides, including the highly studied D-4F peptide. Thesehave a longer half-life and can be given orally, but the high cost ofmanufacturing and accumulation of D-peptides in tissues may bepreventing these peptides from moving past initial clinical testing.

RNase

RNase has been studied as a therapy for cancer and autoimmune disease.For cancer therapy, both natural (onconase, frog RNase), and recombinanthuman RNase1 resistant to inhibition by cytoplasmic inhibitor (see U.S.Pat. No. 8,569,457) have been reported. In addition, targeting of RNaseto tumor cells by conjugation of cytotoxic RNase (onconase) toanti-tumor antibodies has been reported. See Lui et al., Mol. Cancer13:1186, 2014; Newton et al., Blood 97:528-535, 2001.

RNase therapy has also been studied in a mouse model of cardiovasculardisease. See Simsekyilmaz et al., Circulation 129:598-606, 2014. Theyand others show that extracellular RNA accumulates at sites of vascularinjury and that extracellular RNA causes production of inflammatorycytokines. See Fischer et al., Thromb. Haemost. 108:730-741, 2012. RNasetherapy reduced neointima formation in a mouse model of acceleratedcardiovascular disease, reduced plaque macrophage content, and inhibitedleukocyte recruitment to injured carotid arteries in vivo. SeeSimsekyilmaz et al., supra.

RNase therapy has also been studied in models of acute stroke, where itwas found to reduce infarction size. See Walberer et al., Curr.Neurovasc. Res. 6:12-19, 2009. Thus systemic treatment with RNase 1rescued mice from arterial thrombotic occlusion to limit cerebral edemaand to serve as a potent anti-inflammatory regimen in vivo. In theseRNase therapy studies, the RNase was given by continuous infusion usingosmotic minipumps implanted subcutaneously because the half-life ofRNase 1 is very short.

RNase therapy has also been studied in a mouse model of systemic lupuserythematosus (SLE). See Sun et al., J. Immunol. 190:2536-2543, 2013.Overexpression of TLR7, an RNA sensor, causes a lupus-like disease withautoantibodies, kidney disease, and early mortality. Crossing these micewith mice that overexpress RNase A as a transgene resulted in progenywith increased survival, reduced lymphocyte activation, reduced kidneydeposits of IgG and C3, and reduced hepatic inflammation and necrosis.

Extracellular single stranded viral RNA caused widespreadneurodegeneration after intrathecal administration to mice, and theneuronal damage was mediated by TLR7. See Lehmann et al., J. Immunol.189: 1448-58, 2012.

RNase-Ig wherein human RNase 1 is fused to a mutated human IgG1 Fcdomain comprising p238s and p331s mutations (see U.S. Pat. No.8,937,157) is in clinical development by Resolve Therapeutics inpatients with systemic lupus erythematosus (SLE).

Paraoxonase

Human Paraoxonase 1 (PON1) is a lipolactonase with efficient esteraseactivity and capable of hydrolyzing organophosphates. PON1 prevents LDLand cell membrane oxidation and is considered to be atheroprotective.PON1 is exclusively associated with HDL and contributes to theantioxidative function of HDL. See, e.g., Mackness et al., Gene567:12-21, 2015. Reductions in HDL-PON1 activity are present in a widevariety of inflammatory diseases where loss of PON1 activity leads todysfunctional HDL which can promote inflammation and atherosclerosis.See, e.g., Eren et al., Cholesterol. 792090 doi 10.1155/2013/792090,2013. PON1 activity is also decreased in patients with Alzheimer'sdisease and other dementias, suggesting a possible neuroprotective roleof PON1. See Menini et al., Redox Rep. 19:49-58, 2014.

PON1 has shown protective activity in multiple animal models.Overexpression of human PON1 inhibited the development ofatherosclerosis in mice with combined leptin and LDL receptordeficiency, a model of metabolic syndrome. See Mackness et al.,Arterioscler. Thromb. Vasc. Biol. 26:1545-50, 2006.

In another study, injection of recombinant PON1 to mice prior toSTZ-induced diabetes resulted in reduced incidence of diabetes andhigher serum insulin levels. Addition of HDL simultaneously with PON1had an additive effect on insulin secretion. See Koren-Gluser et al.,Atherosclerosis 219:510-518, 2011.

In another study, a PON1 fusion protein containing a proteintransduction domain (PTD) was used to transduce PON1 into cells andtissues. PON1 transduction protected microglial cells in vitro fromoxidative stress-induced inflammatory responses and protected againstdopaminergic neuronal cell death in a Parkinsons disease model. See Kimet al., Biomaterials 64:45-56, 2015.

In another study, recombinant PON1 was administered to mice where therewas a significant reduction in cholesterol mass and an inhibition in thecholesterol biosynthesis rate, effects that could probably lead toattenuation of atherosclerosis. See Rosenblat et al., Biofactors37:462-467, 2011.

In another study, mice were given recombinant adenovirus PON1 or PON3and either was shown to protect against CC1(4)-induced liver injury.Overexpression of either human PON1 or human PON3 reduced hepaticoxidative stress and strengthened the antioxidant capabilities in theliver. See Peng et al., Toxicol. Lett. 193:159-166, 2010.

In another study, PON1 was fused to the C-terminus of an Fc domain, andexpressed as a bispecific molecule using an antibody to human insulinreceptor (HIR). This molecule, termed HIRMAb-PON1, was stable afterexpression in CHO cells, and was shown in Rhesus monkeys to have a highblood brain barrier permeation but was rapidly cleared by the liver. SeeBoado et al., Biotechnol. Bioeng. 108: 186-196, 2011.

Platelet-Activating Factor Acetylhydrolase

Platelet-activating factor acetylhydrolase (PAF-AH) is an LDL andHDL-associated enzyme that hydrolyzes short chain acyl groups ofphospholipids such as platelet-activating factor and oxidizedphospholipids to reduce their inflammatory properties. See Watson etal., J. Clin. Invest. 95:774-782, 1995; Stafforini, Cardiovasc. DrugsTher. 23:73-83, 2009. Therapy with PAF-AH through adenovirus-mediatedgene delivery has been reported to ameliorate proteinuria andglomerulosclerosis in a rat model. See Iso-O et al., Molecular Therapy13:118-126, 2006. PAF-AH also enhanced liver recovery after paracetamolintoxication in the rat, and PAF is associated with liver toxicity fromhigh doses of acetaminophen. See Grypioti et al., Dig. Dis. Sci.52:2580-2590, 2007; Grypioti et al., Dig. Dis. Sci. 53:1054-1062, 2008.A mutation in PAF-AH that causes a loss of function is present in 4% ofJapanese, and PAF-AH was found to be an independent risk factor forcardiovascular disease and stroke in these individuals. See Blankenberget al., J. Lipid Res. 44:1381-1386, 2003. Recombinant PAF-AH was testedin phase III clinical trials in patients with acute respiratory distresssyndrome (ARDS) and in patients with sepsis. See Karabina et al.,Biochim. Biophys. Acta 1761:1351-1358, 2006.

Cholesteryl Ester Transfer Protein

Cholesteryl ester transfer protein (CETP) transports cholesteryl esterfrom high-density lipoproteins (HDL) to low density and very low densitylipoproteins (LDL and VLDL). Many CETP inhibitors have been developedand tested in clinical trials. Torecetrapib was the first CETP inhibitorto advance to late stage clinical trials, and showed a significanteffect on plasma lipoprotein levels, raising antiatherogenic HDLcholesterol levels while lowering proatherogenic LDL cholesterol levels.Torecetrapib binds deeply within CETP and shifts the bound cholesterylester in the N-terminal pocket of the hydrophobic tunnel and displacesphospholipid from the pocket. See Liu et al., J. Biol. Chem.287:37321-37329, 2012. Initial hopes that CETP inhibitors would beuseful for therapy of cardiovascular disease have not been fulfilled;four inhibitors have reached late stage clinical trials but have failedto show a reduction in cardiovascular events. See Kosmas et al.,Clinical Medical Insights: Cardiology 2016: 10 37-42 doi:10.4137/CMC.S32667).

Alternative views of CETP inhibitors and cardiovascular disease haveemerged. See, e.g., Miller, F100Research 3:124, 2014. There is mountingevidence for a protective role of CETP. For example, multiple studies inman now show that cardiovascular disease is related inversely to CETPlevels. In addition, CETP alleles that have reduced hepatic secretionare associated with increased risk of myocardial infarction. See Miller,supra. The original idea, that CETP inhibitors increase HDL cholesterollevels and would therefore be beneficial in reducing cardiovasculardisease, may not be correct. It is likely that HDL cholesterol isbeneficial because of its lipid transport function, and theCETP-mediated transfer of cholesteryl ester from HDL to LDL and VLDL isan important component of this function.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a fusion polypeptidecomprising, from an amino-terminal position to a carboxyl-terminalposition, ApoA1-L1-D, where ApoA1 is a first polypeptide segment havingcholesterol efflux activity and which is selected from (i) a polypeptidecomprising an amino acid sequence having at least 90% or at least 95%identity with amino acid residues 19-267, 25-267, or 1-267 of SEQ IDNO:2 and (ii) an ApoA-1 mimetic; L1 is a first polypeptide linker; and Dis a dimerizing domain. In certain embodiments, L1 comprises at leasttwo amino acid residues, at least three amino acid residues, or at least16 amino acid residues. For example, in particular variations, L1consists of from two to 60 amino acid residues, from three to 60 aminoacid residues, from five to 40 amino acid residues, from 15 to 40 aminoacid residues, or from 16 to 36 amino acid residues. In more specificvariations, L1 consists of 16 amino acid residues, 21 amino acidresidues, 26 amino acid residues, 31 amino acid residues, or 36 aminoacid residues; in some such embodiments, L1 has the amino acid sequenceshown in residues 268-283 of SEQ ID NO:22, residues 268-288 of SEQ IDNO:26, residues 268-293 of SEQ ID NO:2, SEQ ID NO:54, or residues268-303 of SEQ ID NO:24. In certain embodiments, the first polypeptidesegment comprises the amino acid sequence shown in residues 19-267 or25-267 of SEQ ID NO:2.

In some embodiments of a fusion polypeptide as above, the firstpolypeptide segment comprises an amino acid sequence having at least 90%or at least 95% identity with amino acid residues 19-267, 25-267, or1-267 of SEQ ID NO:2, where the first polypeptide segment comprises atleast one amino acid substitution (relative to SEQ ID NO:2) selectedfrom (a) valine at the amino acid position corresponding to position 180of SEQ ID NO:2 replaced with glutamate or lysine (“V156[E/1(]”); (b)tyrosine at the amino acid position corresponding to position 216 of SEQID NO:2 replaced with serine, glutamine, asparagine, histidine, orphenylalanine (“Y192[S/Q/N/H/F]”); (c) methionine at the amino acidposition corresponding to position 110 of SEQ ID NO:2 replaced withleucine, isoleucine, or valine (“M86[L/I/V]”); (d) methionine at theamino acid position corresponding to position 136 of SEQ ID NO:2replaced with leucine, isoleucine, or valine (“M112[L/I/V]”); (e)methionine at the amino acid position corresponding to position 172 ofSEQ ID NO:2 replaced with leucine, isoleucine, or valine(“M148[L/I/V]”); (f) tryptophan at the amino acid position correspondingto position 32 of SEQ ID NO:2 replaced with phenylalanine (“W8F”); (g)tryptophan at the amino acid position corresponding to position 74 ofSEQ ID NO:2 replaced with phenylalanine (“W50F”); (h) tryptophan at theamino acid position corresponding to position 96 of SEQ ID NO:2 replacedwith phenylalanine (“W72F”); and (i) tryptophan at the amino acidposition corresponding to position 132 of SEQ ID NO:2 replaced withphenylalanine (“W132F”). In some such embodiments, the first polypeptidesegment comprises the V156[E/K] or Y192[S/Q/N/H/F] substitution, andoptionally further comprises at least one substitution selected fromM86[L/I/V], M112[L/I/V], and M148[L/I/V]. In another variation, thefirst polypeptide segment comprises the both the V156[E/K] andY192[S/Q/N/H/F] substitutions, and optionally further comprises at leastone substitution selected from M86[L/I/V], M112[L/I/V], and M148[L/I/V].In another variation, the first polypeptide segment comprises the W8F,W50F, W72F, and W132F substitutions. In certain embodiments, the firstpolypeptide segment comprises one or more of the substitutions selectedfrom V156[E/K], Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V],W8F, W50F, W72F, and W132F (e.g., V156[E/K] or Y192[S/Q/N/H/F] andoptionally at least one of M86[L/I/V], M112[L/I/V], and M148[L/I/V];both V156[E/K] and Y192[S/Q/N/H/F] and optionally at least one ofM86[L/I/V], M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F,W72F, and W132F) but comprises an amino acid sequence that is otherwise100% identical to residues 19-267, 25-267, or 1-267 of SEQ ID NO:2. Insome variations, the first polypeptide segment comprises an amino acidsequence having at least 90% or at least 95% identity with amino acidresidues 19-267, 25-267, or 1-267 of SEQ ID NO:2, where the firstpolypeptide segment comprises the specific V156[E/K] and/orY192[S/Q/N/H/F] substitution(s) of any one of variant combinationsA1-A17 as shown in Table 3 herein; in some such embodiments, the firstpolypeptide segment comprises an amino acid sequence that is otherwise100% identical to residues 19-267, 25-267, or 1-267 of SEQ ID NO:2.

In some embodiments of a fusion polypeptide as above, D is animmunoglobulin heavy chain constant region such as, for example, animmunoglobulin Fc region. In certain embodiments where the dimerizingdomain is an immunoglobulin Fc region, the Fc region is a human Fcregion such as, e.g., a human Fc variant comprising one or more aminoacid substitutions relative to the wild-type human sequence.Particularly suitable Fc regions include human γ1 and γ3 Fc regions. Insome variations, the Fc region is a human γ1 Fc variant in which Euresidue C220 is replaced by serine; in some such embodiments Eu residuesC226 and C229 are each replaced by serine, and/or Eu residue P238 isreplaced by serine. In further variations comprising an Fc region asabove, the Fc region is a human γ1 Fc variant in which Eu residue P331is replaced by serine. Fc variants may include an amino acidsubstitution that reduces glycosylation relative to the wild-type humansequence; in some such embodiments, Eu residue N297 is replaced withanother amino acid. In further variations comprising an Fc region asabove, the Fc region is an Fc variant comprising an amino acidsubstitution that increases or reduces binding affinity for an Fcreceptor (e.g., an amino acid substitution that increases or reducesbinding affinity for at least one of FcγRI, FcγRH, and FcγRIII). Incertain embodiments, an Fc variant includes an amino acid substitutionthat increases or reduces binding affinity for the neonatal Fc receptor(FcRn). Suitable Fc regions include (i) an Fc region comprising theamino acid sequence shown in residues 294-525 or 294-524 of SEQ ID NO:2and (ii) an Fc region comprising the amino acid sequence shown inresidues 294-525 or 294-524 of SEQ ID NO:13.

In certain embodiments of a fusion polypeptide as above, the fusionpolypeptide comprises an amino acid sequence having at least 90% or atleast 95% identity with (i) residues 19-525, 19-524, 25-525, or 25-524of SEQ ID NO:2, (ii) residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:13, (iii) residues 19-501, 19-500, 25-501, or 25-500 of SEQ IDNO:20, (iv) residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22,(v) residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26, or (vi)residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24. In morespecific variations, the fusion polypeptide comprises the amino acidsequence shown in (i) residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:2, (ii) residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:13, (iii) residues 19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20,(iv) residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22, (v)residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26, or (vi)residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24. In someembodiments, the fusion polypeptide comprises an amino acid sequencehaving at least 90% or at least 95% identity with (i) residues 19-525,19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii) residues 19-525, 19-524,25-525, or 25-524 of SEQ ID NO:13, (iii) residues 19-501, 19-500,25-501, or 25-500 of SEQ ID NO:20, (iv) residues 19-515, 19-514, 25-515,or 25-514 of SEQ ID NO:22, (v) residues 19-520, 19-519, 25-520, or25-519 of SEQ ID NO:26, or (vi) residues 19-535, 19-534, 25-535, or25-534 of SEQ ID NO:24, where the fusion polypeptide comprises one ormore amino acid substitutions in the first polypeptide segment selectedfrom V156[E/K], Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V],W8F, W50F, W72F, and W132F as described herein (e.g., V156[E/K] orY192[S/Q/N/H/F] and optionally at least one of M86[L/I/V], M112[L/I/V],and M148[L/I/V]; both V156[E/K] and Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; or all four ofW8F, W50F, W72F, and W132F); in some such embodiments, the fusionpolypeptide comprises an amino acid sequence that is otherwise 100%identical to (i) residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:2, (ii) residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13,(iii) residues 19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20, (iv)residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22, (v) residues19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26, or (vi) residues19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24.

In some embodiments of a fusion polypeptide as above, the fusionpolypeptide further includes a second polypeptide segment locatedcarboxyl-terminal to the dimerizing domain. In particular variations,the second polypeptide segment is an RNase, a paraoxonase, aplatelet-activating factor acetylhydrolase (PAF-AH), a cholesterol estertransfer protein (CETP), a lecithin-cholesterol acyltransferase (LCAT),or a polypeptide that specifically binds to amyloid beta (Aβ) such as,e.g., an Aβ-specific scFv. A fusion polypeptide comprising a secondpolypeptide segment as above may be represented by the formulaApoA1-L1-D-L2-P (from an amino-terminal position to a carboxyl-terminalposition), where ApoA1, L1, and D are each defined as above, where L2 isa second polypeptide linker and is optionally present, and where P isthe second polypeptide segment. In some embodiments of a fusionpolypeptide where L2 is present, L2 has the amino acid sequence shown inresidues 526-543 of SEQ ID NO:4.

In another aspect, the present invention provides a fusion polypeptidecomprising a first polypeptide segment having cholesterol effluxactivity and which is selected from (i) a polypeptide comprising anamino acid sequence having at least 90% or at least 95% identity withamino acid residues 19-267 or 25-267 of SEQ ID NO:2 and (ii) an ApoA-1mimetic, and a second polypeptide segment located carboxyl-terminal tothe first polypeptide segment, where the second polypeptide segment isselected from an RNase, a paraoxonase, a platelet-activating factoracetylhydrolase (PAF-AH), a cholesterol ester transfer protein (CETP), alecithin-cholesterol acyltransferase (LCAT), and a polypeptide thatspecifically binds to amyloid beta such as, e.g., an Aβ-specific scFv.In some embodiments, the first polypeptide segment has the amino acidsequence shown in residues 19-267 or 25-267 of SEQ ID NO:2. In somevariations, the fusion polypeptide further includes a linker polypeptidelocated carboxyl-terminal to the first polypeptide segment andamino-terminal to the second polypeptide segment. In some embodiments,the fusion polypeptide further includes a dimerizing domain.

In some embodiments of a fusion polypeptide as above comprising an RNaseas a second polypeptide segment, the RNase is human RNAse 1 or afunctional variant or fragment thereof. In certain embodiments, theRNase has at least 90% or at least 95% identity with amino acid residues544-675 or 548-675 of SEQ ID NO:4. In a specific variation, the RNasehas the amino acid sequence shown in residues 544-675 or 548-675 of SEQID NO:4. In particular embodiments of a fusion polypeptide comprising anRNase and having the formula ApoA1-L1-D-L2-P as above, the fusionpolypeptide comprises an amino acid sequence having at least 90% or atleast 95% identity with (i) residues 19-675 or 25-675 of SEQ ID NO:4,(ii) residues 19-675 or 25-675 of SEQ ID NO:14, (iii) residues 19-671 or25-671 of SEQ ID NO:58, or (iv) residues 19-671 or 25-671 of SEQ IDNO:59; in some such embodiments, the fusion polypeptide comprises theamino acid sequence shown in (i) residues 19-675 or 25-675 of SEQ IDNO:4, (ii) residues 19-675 or 25-675 of SEQ ID NO:14, (iii) residues19-671 or 25-671 of SEQ ID NO:58, or (iv) residues 19-671 or 25-671 ofSEQ ID NO:59. In some embodiments, the fusion polypeptide comprises anamino acid sequence having at least 90% or at least 95% identity with(i) residues 19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or25-675 of SEQ ID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58,or (iv) residues 19-671 or 25-671 of SEQ ID NO:59, where the fusionpolypeptide comprises one or more amino acid substitutions in the firstpolypeptide segment selected from V156[E/K], Y192[S/Q/N/H/F],M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F, and W132F asdescribed herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; both V156[E/K]and Y192[S/Q/N/H/F] and optionally at least one of M86[L/I/V],M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F, W72F, andW132F); in some such embodiments, the fusion polypeptide comprises anamino acid sequence that is otherwise 100% identical to (i) residues19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or 25-675 of SEQID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or (iv)residues 19-671 or 25-671 of SEQ ID NO:59.

In some embodiments of a fusion polypeptide as above comprising aparaoxonase as a second polypeptide segment, the paraoxonase is humanparaoxonase 1 (PON1) or a functional variant thereof. In certainembodiments, the paraoxonase has at least 90% or at least 95% identitywith amino acid residues 16-355 of SEQ ID NO:12, amino acid residues16-355 of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44.In specific variations, the paraoxonase comprises the amino acidsequence shown in residues 16-355 of SEQ ID NO:12, residues 16-355 ofSEQ ID NO:42, or residues 16-355 of SEQ ID NO:44. In particularembodiments of a fusion polypeptide comprising an paraoxonase and havingthe formula ApoA1-L1-D-L2-P as above, the fusion polypeptide comprisesan amino acid sequence having at least 90% or at least 95% identity with(i) residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46,or (iv) residues 19-883 or 25-883 of SEQ ID NO:48; in some suchembodiments, the fusion polypeptide comprises the amino acid sequenceshown in (i) residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues19-873 or 25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQID NO:46, or (iv) residues 19-883 or 25-883 of SEQ ID NO:48. In someembodiments of a fusion polypeptide as above comprising a paraoxonase asa second polypeptide segment, the second polypeptide segment comprisesan amino acid sequence having at least 90% or at least 95% identity withamino acid residues 16-355 of SEQ ID NO:12, amino acid residues 16-355of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44, where(a) tyrosine at the amino acid position corresponding to position 185 ofSEQ ID NO:12, SEQ ID NO:42, or SEQ ID NO:44 is replaced with histidine,glutamine, or serine (“Y185[H/Q/S]” substitution) and/or phenylalanineat the amino acid position corresponding to position 293 of SEQ IDNO:12, SEQ ID NO:42, or SEQ ID NO:44 is replaced with histidine,glutamine, or asparagine (“F293[H/Q/N]” substitution); in some suchembodiments, the second polypeptide segment comprises the Y185[H/Q/S]substitution and/or the F293[H/Q/S] substitution but comprises an aminoacid sequence that is otherwise 100% identical to residues 16-355 of SEQID NO:12, residues 16-355 of SEQ ID NO:42, or residues 16-355 of SEQ IDNO:44. In some variations, the second polypeptide segment comprises anamino acid sequence having at least 90% or at least 95% identity withamino acid residues 16-355 of SEQ ID NO:12, amino acid residues 16-355of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44, wherethe second polypeptide segment comprises the specific Y185[H/Q/S] and/orF293[H/Q/N] substitution(s) of any one of variant combinations P1-P15 asshown in Table 4 herein; in some such embodiments, the secondpolypeptide segment comprises an amino acid sequence that is otherwise100% identical to residues 16-355 of SEQ ID NO:12, residues 16-355 ofSEQ ID NO:42, or residues 16-355 of SEQ ID NO:44. In some embodiments,the fusion polypeptide comprises an amino acid sequence having at least90% or at least 95% identity with (i) residues 19-883 or 25-883 of SEQID NO:28, (ii) residues 19-873 or 25-873 of SEQ ID NO:38, (iii) residues19-883 or 25-883 of SEQ ID NO:46, or (iv) residues 19-883 or 25-883 ofSEQ ID NO:48, where the fusion polypeptide comprises (A) at least oneamino acid substitution in the first polypeptide segment selected fromV156[E/K], Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F,W50F, W72F, and W132F as described herein (e.g., V156[E/K] orY192[S/Q/N/H/F] and optionally at least one of M86[L/I/V], M112[L/I/V],and M148[L/I/V]; both V156[E/K] and Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; or all four ofW8F, W50F, W72F, and W132F), and/or (B) at least one substitution in thesecond polypeptide segment selected from Y185[H/Q/S] and F293[H/Q/N] asdescribed herein; in some such embodiments, the fusion polypeptidecomprises an amino acid sequence that is otherwise 100% identical to (i)residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46,or (iv) residues 19-883 or 25-883 of SEQ ID NO:48; in particularvariations, the fusion polypeptide comprises substitutions of both (A)and (B) as above (e.g., V156[E/K] and/or Y192[S/Q/N/H/F] and optionallyat least one of M86[L/I/V], M112[L/I/V], and M148[L/I/V] in the firstpolypeptide segment and Y185[H/Q/S] and optionally F293[H/Q/N] in thesecond polypeptide segment).

In some embodiments of a fusion polypeptide as above comprising aplatelet-activating factor acetylhydrolase (PAF-AH) as a secondpolypeptide segment, the platelet-activating factor acetylhydrolase is ahuman PAF-AH or a functional variant thereof. In certain embodiments,the platelet-activating factor acetylhydrolase has at least 90% or atleast 95% identity with amino acid residues 22-441 of SEQ ID NO:32. In aspecific variation, the platelet-activating factor acetylhydrolasecomprises the amino acid sequence shown in residues 22-441 of SEQ IDNO:32. In particular embodiments of a fusion polypeptide comprising aplatelet-activating factor acetylhydrolase and having the formulaApoA1-L1-D-L2-P as above, the fusion polypeptide comprises an amino acidsequence having at least 90% or at least 95% identity with residues19-963 or 25-963 of SEQ ID NO:34; in some such embodiments, the fusionpolypeptide comprises the amino acid sequence shown in residues 19-963or 25-963 of SEQ ID NO:34. In some embodiments, the fusion polypeptidecomprises an amino acid sequence having at least 90% or at least 95%identity with residues 19-963 or 25-963 of SEQ ID NO:34, where thefusion polypeptide comprises one or more amino acid substitutions in thefirst polypeptide segment selected from V156[E/K], Y192[S/Q/N/H/F],M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F, and W132F asdescribed herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; both V156[E/K]and Y192[S/Q/N/H/F] and optionally at least one of M86[L/I/V],M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F, W72F, andW132F); in some such embodiments, the fusion polypeptide comprises anamino acid sequence that is otherwise 100% identical to residues 19-963or 25-963 of SEQ ID NO:34.

In some embodiments of a fusion polypeptide as above comprising acholesterol ester transfer protein (CETP) as a second polypeptidesegment, the cholesterol ester transfer protein is human CETP or afunctional variant thereof. In certain embodiments, the cholesterolester transfer protein has at least 90% or at least 95% identity withamino acid residues 18-493 of SEQ ID NO:30. In a specific variation, thecholesterol ester transfer protein comprises the amino acid sequenceshown in residues 18-493 of SEQ ID NO:30. In particular embodiments of afusion polypeptide comprising a cholesterol ester transfer protein andhaving the formula ApoA1-L1-D-L2-P as above, the fusion polypeptidecomprises an amino acid sequence having at least 90% or at least 95%identity with residues 19-1019 or 25-1019 of SEQ ID NO:40; in some suchembodiments, the fusion polypeptide comprises the amino acid sequenceshown in residues 19-1019 or 25-1019 of SEQ ID NO:40. In someembodiments, the fusion polypeptide comprises an amino acid sequencehaving at least 90% or at least 95% identity with residues 19-1019 or25-1019 of SEQ ID NO:40, where the fusion polypeptide comprises one ormore amino acid substitutions in the first polypeptide segment selectedfrom V156[E/K], Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V],W8F, W50F, W72F, and W132F as described herein (e.g., V156[E/K] orY192[S/Q/N/H/F] and optionally at least one of M86[L/I/V], M112[L/I/V],and M148[L/I/V]; both V156[E/K] and Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; or all four ofW8F, W50F, W72F, and W132F); in some such embodiments, the fusionpolypeptide comprises an amino acid sequence that is otherwise 100%identical to residues 19-1019 or 25-1019 of SEQ ID NO:40.

In certain embodiments of a fusion polypeptide as above, the fusionpolypeptide is linked to a myeloperoxidase (MPO) inhibitor.

In another aspect, the present invention provides a dimeric proteincomprising a first fusion polypeptide and a second fusion polypeptide,where each of said first and second fusion polypeptides is a fusionpolypeptide comprising a dimerizing domain, as described above.

In another aspect, the present invention provides a polynucleotideencoding a fusion polypeptide as described above.

In still another aspect, the present invention provides an expressionvector comprising the following operably linked elements: atranscription promoter, a DNA segment encoding a fusion polypeptide asdescribed above, and a transcription terminator. Also provided is acultured cell into which has been introduced an expression vector asabove, wherein the cell expresses the DNA segment.

In another aspect, the present invention provides a method of making afusion polypeptide. The method generally includes culturing a cell intowhich has been introduced an expression vector as described above, wherethe cell expresses the DNA segment and the encoded fusion polypeptide isproduced, and recovering the fusion polypeptide.

In yet another aspect, the present invention provides a method of makinga dimeric protein. The method generally includes culturing a cell intowhich has been introduced an expression vector as described above, wherethe cell expresses the DNA segment and the encoded fusion polypeptide isproduced as a dimeric protein, and recovering the dimeric protein.

In another aspect, the present invention provides a compositioncomprising a fusion polypeptide as described above and apharmaceutically acceptable carrier.

In another aspect, the present invention provides a compositioncomprising a dimeric protein as described above and a pharmaceuticallyacceptable carrier.

In still another aspect, the present invention provides a method fortreating a cardiovascular disease characterized by atherosclerosis. Themethod generally includes administering to a subject having thecardiovascular disease an effective amount of a fusion polypeptide ordimeric fusion protein as described above. In some embodiments, thecardiovascular disease is selected from the group consisting of coronaryheart disease and stroke. In certain variations, the coronary heartdisease is characterized by acute coronary syndrome.

In another aspect, the present invention provides a method for treatinga neurodegenerative disease. The method generally includes administeringto a subject having the neurodegenerative disease an effective amount ofa fusion polypeptide or dimeric fusion protein as described above. Insome embodiments, the neurodegenerative disease is selected from thegroup consisting of Alzheimer's disease and multiple sclerosis. Incertain embodiments, the neurodegenerative disease is characterized bydementia; in some such variations, the neurodegenerative disease isAlzheimer's disease.

In another aspect, the present invention provides a method for treatinga disease characterized by amyloid deposit. The method generallyincludes administering to a subject having the disease characterized byamyloid deposit an effective amount of a fusion polypeptide or dimericfusion protein as described above. In some embodiments, the disease isAlzheimer's disease.

In another aspect, the present invention provides a method for treatingan autoimmune disease. The method generally includes administering to asubject having the autoimmune disease an effective amount of a fusionpolypeptide or dimeric fusion protein as described above. In someembodiments, the autoimmune disease is selected from the groupconsisting of rheumatoid arthritis, systemic lupus erythematosus,multiple sclerosis, and type 1 diabetes.

In yet another aspect, the present invention provides a method fortreating an inflammatory disease. The method generally includesadministering to a subject having the inflammatory disease an effectiveamount of a fusion polypeptide or dimeric fusion protein as describedabove. In some embodiments, the inflammatory disease is selected fromthe group consisting of rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, type 1 diabetes, type 2 diabetes,obesity, non-alcoholic steatohepatitis, coronary heart disease, andstroke. In other embodiments, the inflammatory disease is aninflammatory lung disease such as, for example, asthma, chronicobstructive pulmonary disease (COPD), bronchiectasis, idiopathicpulmonary fibrosis, hyperoxia, hypoxia, or acute respiratory distresssyndrome.

In still another aspect, the present invention provides a method fortreating an infectious disease. The method generally includesadministering to a subject having the infectious disease an effectiveamount of a fusion polypeptide or dimeric fusion protein as describedabove. In certain embodiments, the infectious disease is characterizedby a bacterial infection; in some such embodiments, the bacterialinfection is a Pseudomonas aeruginosa infection.

In another aspect, the present invention provides a method for treatingnephrotic syndrome (NS). The method generally includes administering toa subject having nephrotic syndrome an effective amount of a fusionpolypeptide or dimeric fusion protein as described above. In specificvariations, the subject's nephrotic syndrome is associated with adisease selected from the group consisting of a primary kidney disease(e g, minimal-change nephropathy, focal glomerulosclerosis, membranousnephropathy, or IgA nephropathy), amyloidosis, systemic lupuserythematosus, type 1 diabetes, and type 2 diabetes.

In yet another aspect, the present invention provides a method fortreating exposure to sulfur mustard gas or to an organophosphate. Themethod generally includes administering to a subject exposed to thesulfur mustard gas or to the organophosphate an effective amount of afusion polypeptide or dimeric fusion protein as described above.

In still another aspect, the present invention provides a method fortreating cancer. The method generally includes administering to asubject having cancer an effective amount of a fusion polypeptide ordimeric fusion protein as described above. In some embodiments, thecancer is selected from the group consisting of malignant melanoma,renal cell carcinoma, non-small cell lung cancer, bladder cancer, andhead and neck cancer. In certain variations, the cancer treatment is acombination therapy. In some combination therapy embodiments, thecombination therapy includes a non-ApoA1-mediated immunomodulatorytherapy such as, e.g., an immunomodulatory therapy comprising ananti-PD-1/PD-L1 therapy, an anti-CTLA-4 therapy, or both. In othercombination therapy embodiments, the combination therapy includesradiation therapy or chemotherapy. In some combination therapyembodiments, the combination therapy includes a targeted therapy; insome such embodiments, the targeted therapy includes (i) a therapeuticmonoclonal antibody targeting a specific cell-surface or extracellularantigen (e.g., VEGF, EGFR, CTLA-4, PD-1, or PD-L1) or (ii) a smallmolecule targeting an intracellular protein such as, for example, anintracellular enzyme (e.g., a proteasome, a tyrosine kinase, acyclin-dependent kinase, serine/threonine-protein kinase B-Raf (BRAF),or a MEK kinase).

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The terms “amino-terminal” (or “N-terminal”) and “carboxyl-terminal” (or“C-terminal”) are used herein to denote positions within polypeptides.Where the context allows, these terms are used with reference to aparticular sequence or portion of a polypeptide to denote proximity orrelative position. For example, a certain sequence positionedcarboxyl-terminal to a reference sequence within a polypeptide islocated proximal to the carboxyl terminus of the reference sequence, butis not necessarily at the carboxyl terminus of the complete polypeptide.

The terms “polynucleotide” and “nucleic acid” are used synonymouslyherein and refer to a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. It will berecognized by those skilled in the art that the two strands of adouble-stranded polynucleotide may differ slightly in length and thatthe ends thereof may be staggered as a result of enzymatic cleavage;thus all nucleotides within a double-stranded polynucleotide moleculemay not be paired. Such unpaired ends will in general not exceed 20 ntin length.

A “segment” is a portion of a larger molecule (e.g., polynucleotide orpolypeptide) having specified attributes. For example, a DNA segmentencoding a specified polypeptide is a portion of a longer DNA molecule,such as a plasmid or plasmid fragment that, when read from the 5′ to the3′ direction, encodes the sequence of amino acids of the specifiedpolypeptide. Also, in the context of a fusion polypeptide in accordancewith the present invention, a polypeptide segment “having cholesterolefflux activity” and “comprising an amino acid sequence having at least90% or at least 95% identity with amino acid residue 19-267 or 25-267 ofSEQ ID NO:2” is a portion of the longer polypeptide fusion moleculethat, in addition to the specified polypeptide segment havingcholesterol efflux activity, includes other polypeptide segments (e.g.,linker(s), dimerizing domain) as described herein.

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

“Operably linked” means that two or more entities are joined togethersuch that they function in concert for their intended purposes. Whenreferring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

The term “recombinant” when used with reference, e.g., to a cell,nucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein, or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,under-expressed or not expressed at all. By the term “recombinantnucleic acid” herein is meant nucleic acid, originally formed in vitro,in general, by the manipulation of nucleic acid using, e.g., polymerasesand endonucleases, in a form not normally found in nature. In thismanner, operable linkage of different sequences is achieved. Thus anisolated nucleic acid, in a linear form, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, areboth considered recombinant for the purposes disclosed herein. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.,using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes disclosed herein.Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e., through the expression of a recombinant nucleic acidas depicted above.

The term “heterologous,” when used with reference to portions of anucleic acid, indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, “heterologous,” when used in reference to portions of aprotein, indicates that the protein comprises two or more subsequencesthat are not found in the same relationship to each other in nature(e.g., two or segments of a fusion polypeptide).

An “immunoglobulin” is a serum protein which functions as an antibody ina vertebrate organism. Five classes of “immunoglobulin,” or antibody,protein (IgG, IgA, IgM, IgD, and IgE) have been identified in highervertebrates. IgG comprises the major class; it normally exists as thesecond most abundant protein found in plasma. In humans, IgG consists offour subclasses, designated IgG1, IgG2, IgG3, and IgG4. The heavy chainconstant regions of the IgG class are identified with the Greek symbolγ. For example, immunoglobulins of the IgG1 subclass contain a yl heavychain constant region. Each immunoglobulin heavy chain possesses aconstant region that consists of constant region protein domains (CH1,hinge, CH2, and CH3) that are essentially invariant for a given subclassin a species. DNA sequences encoding human and non-human immunoglobulinchains are known in the art. See, e.g., Ellison et al., DNA 1:11-18,1981; Ellison et al., Nuc. Acids Res. 10:4071-4079, 1982; Kenten et al.,Proc. Natl. Acad. Sci. USA 79:6661-6665, 1982; Seno et al., Nuc. AcidsRes. 11:719-726, 1983; Riechmann et al., Nature 332:323-327, 1988;Amster et al., Nuc. Acids Res. 8:2055-2065, 1980; Rusconi and Kohler,Nature 314:330-334, 1985; Boss et al., Nuc. Acids Res. 12:3791-3806,1984; Bothwell et al., Nature 298:380-382, 1982; van der Loo et al.,Immunogenetics 42:333-341, 1995; Karlin et al., J. Mol. Evol.22:195-208, 1985; Kindsvogel et al., DNA 1:335-343, 1982; Breiner etal., Gene 18:165-174, 1982; Kondo et al., Eur. J. Immunol. 23:245-249,1993; and GenBank Accession No. J00228. For a review of immunoglobulinstructure and function, see Putnam, The Plasma Proteins, Vol V, AcademicPress, Inc., 49-140, 1987; and Padlan, Mol. Immunol. 31:169-217, 1994.

An “immunoglobulin hinge” is that portion of an immunoglobulin heavychain connecting the CH1 and CH2 domains. The hinge region of human γ1corresponds approximately to Eu residues 216-230.

The terms “Fc fragment,” “Fc region,” or “Fc domain,” as used herein,are synonymous and refer to the portion of an immunoglobulin that isresponsible for binding to antibody receptors on cells and the C1qcomponent of complement (in the absence of any amino acid changes,relative to the naturally occurring sequence, to remove such bindingactivity). Fc stands for “fragment crystalline,” the fragment of anantibody that will readily form a protein crystal. Distinct proteinfragments, which were originally described by proteolytic digestion, candefine the overall general structure of an immunoglobulin protein. Asoriginally defined in the literature, the Fc fragment consists of thedisulfide-linked heavy chain hinge regions, CH2, and CH3 domains. Asused herein, the term also refers to a single chain consisting of CH3,CH2, and at least a portion of the hinge sufficient to form adisulfide-linked dimer with a second such chain. As used herein, theterm Fc region further includes variants of naturally occurringsequences, where the variants are capable of forming dimers andincluding such variants that have increased or decreased Fcreceptor-binding or complement-binding activity.

“Dimerizing domain,” as used herein, refers to a polypeptide havingaffinity for a second polypeptide, such that the two polypeptidesassociate under physiological conditions to form a dimer. Typically, thesecond polypeptide is the same polypeptide, although in some variationsthe second polypeptide is different. The polypeptides may interact witheach other through covalent and/or non-covalent association(s). Examplesof dimerizing domains include an Fc region; a hinge region; a CH3domain; a CH4 domain; a CH1 or CL domain; a leucine zipper domain (e.g.,a jun/fos leucine zipper domain, see, e.g., Kostelney et al., J.Immunol., 148:1547-1553, 1992; or a yeast GCN4 leucine zipper domain);an isoleucine zipper domain; a dimerizing region of a dimerizingcell-surface receptor (e.g., interleukin-8 receptor (IL-8R); or anintegrin heterodimer such as LFA-1 or GPIIIb/IIIa); a dimerizing regionof a secreted, dimerizing ligand (e.g., nerve growth factor (NGF),neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growthfactor (VEGF), or brain-derived neurotrophic factor (BDNF); see, e.g.,Arakawa et al., J. Biol. Chem. 269:27833-27839, 1994, and Radziejewskiet al., Biochem. 32:1350, 1993); and a polypeptide comprising at leastone cysteine residue (e.g., from about one, two, or three to about tencysteine residues) such that disulfide bond(s) can form between thepolypeptide and a second polypeptide comprising at least one cysteineresidue (hereinafter “a synthetic hinge”). A preferred dimerizing domainin accordance with the present invention is an Fc region.

The term “dimer” or “dimeric protein” as used herein, refers to amultimer of two (“first” and “second”) fusion polypeptides as disclosedherein linked together via a dimerizing domain. Unless the contextclearly indicates otherwise, a “dimer” or “dimeric protein” includesreference to such dimerized first and second fusion polypeptides in thecontext of higher order multimers that may form in spherical HDLparticles (e.g., trimers), such as through an interaction of dimerizedfirst and second fusion polypeptides with another ApoA-1 polypeptidethat may be present (e.g., through interaction with a naturallyoccurring, endogenous ApoA-1 protein). The term also includes referenceto dimerized first and second fusion polypeptides in the context ofhigher order multimers that may be created by inclusion of an additionaldimerizing domain in a first or second fusion polypeptide (e.g., a firstfusion polypeptide comprising an immunoglobulin light chain and a secondfusion polypeptide comprising an immunoglobulin heavy chain canheterodimerize via the interaction between the CH1 and CL domains, andtwo such heterodimers may further dimerize via the Fc region of theimmunoglobulin heavy chain, thereby forming a tetramer).

The term “linker” or “polypeptide linker” is used herein to indicate twoor more amino acids joined by peptide bond(s) and linking two discrete,separate polypeptide regions. The linker is typically designed to allowthe separate polypeptide regions to perform their separate functions(such as, e.g., where a dimerizing domain, linked to other polypeptideregions, associates with another, corresponding dimerization domain toform a dimer). The linker can be a portion of a native sequence, avariant thereof, or a synthetic sequence. Linkers are also referred toherein using the abbreviation “L.” The use of a subscript (e.g., “1” or“2”) with “L” is used herein to differentiate among multiple linkerswithin a polypeptide chain, which linkers may be the same or differentwith respect to amino acid sequence.

Unless the context clearly indicates otherwise, reference herein to“ApoA-1” is understood to include naturally occurring ApoA-1polypeptides as well as functional variants, functional fragments, andmimetics thereof. “ApoA1,” Apo A-1,” “apoA-1,” and “apo A-1” are usedherein synonymously with “ApoA-1.”

Unless the context clearly indicates otherwise, reference herein “RNase”(e.g., “RNase 1”), “paraoxonase” (e.g., “PON1”), “platelet-activatingfactor acetylhydrolase” (“PAF-AH”), “cholesterol ester transfer protein”(“CETP”), or “lecithin-cholesterol acyltransferase” (“LCAT”) isunderstood to include naturally occurring polypeptides of any of theforegoing, as well as functional variants and functional fragmentsthereof.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

ApoA-1 fusion polypeptides of the present disclosure may be referred toherein by formulae such as, for example, “ApoA1-L1-D,”“ApoA1-L1-D-L2-P,” “ApoA1-L1-[Fc region],” “ApoA1-L1-D-L2-RNase,”“ApoA1-L1-[Fc region]-L2-RNase1,” “ApoA1-L1-D-L2-paraoxonase,” or“ApoA1-L1-[Fc region]-L2-PON1.” In each such case, unless the contextclearly dictates otherwise, a term referring to a particular segment ofa fusion polypeptide (e.g., “ApoA1,” “D” (for dimerizing domain), “L1”(for a first polypeptide linker), “Fc region,” “RNase,” “paraoxonase,”etc.) is understood to have the meaning ascribed to such term herein andis inclusive of the various embodiments as described herein.

The term “effective amount,” in the context of treatment of a disease byadministration of a soluble fusion polypeptide or dimeric protein to asubject as described herein, refers to an amount of such molecule thatis sufficient to inhibit the occurrence or ameliorate one or moresymptoms of the disease. For example, in the specific context oftreatment of an autoimmune disease by administration of a dimeric ApoA1fusion protein to a subject as described herein, the term “effectiveamount” refers to an amount of such molecule that is sufficient tomodulate an autoimmune response in the subject so as to inhibit theoccurrence or ameliorate one or more symptoms of the autoimmune disease.An effective amount of an agent is administered according to the methodsof the present invention in an “effective regime.” The term “effectiveregime” refers to a combination of amount of the agent beingadministered and dosage frequency adequate to accomplish treatment orprevention of the disease.

The term “patient” or “subject,” in the context of treating a disease ordisorder as described herein, includes mammals such as, for example,humans and other primates. The term also includes domesticated animalssuch as, e.g., cows, hogs, sheep, horses, dogs, and cats.

The term “combination therapy” refers to a therapeutic regimen thatinvolves the provision of at least two distinct therapies to achieve anindicated therapeutic effect. For example, a combination therapy mayinvolve the administration of two or more chemically distinct activeingredients, or agents, for example, a soluble ApoA1 fusion polypeptideor dimeric protein according to the present invention and another agentsuch as, e.g., another anti-inflammatory or immunomodulatory agent.Alternatively, a combination therapy may involve the administration of asoluble ApoA1 fusion polypeptide or dimeric protein according to thepresent invention, alone or in conjunction with another agent, as wellas the delivery of another therapy (e.g., radiation therapy). Thedistinct therapies constituting a combination therapy may be delivered,e.g., as simultaneous, overlapping, or sequential dosing regimens. Inthe context of the administration of two or more chemically distinctagents, it is understood that the active ingredients may be administeredas part of the same composition or as different compositions. Whenadministered as separate compositions, the compositions comprising thedifferent active ingredients may be administered at the same ordifferent times, by the same or different routes, using the same ordifferent dosing regimens, all as the particular context requires and asdetermined by the attending physician.

The term “non-ApoA1-mediated immunomodulatory therapy,” in the contextof treating cancer, means an immunomodulatory therapy that does notspecifically target ApoA-1 or ApoA-1-mediated signaling pathways.

The term “targeted therapy,” in the context of treating cancer, refersto a type of treatment that uses a therapeutic agent to identify andattack a specific type of cancer cell, typically with less harm tonormal cells. In some embodiments, a targeted therapy blocks the actionof an enzyme or other molecule involved in the growth and spread ofcancer cells. In other embodiments, a targeted therapy either helps theimmune system to attack cancer cells or delivers a toxic substancedirectly to cancer cells. In certain variations, a targeted therapy usesa small molecule drug or a monoclonal antibody as a therapeutic agent.

Two amino acid sequences have “100% amino acid sequence identity” if theamino acid residues of the two amino acid sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing amino acid sequences bydetermining optimal alignment are well-known to those of skill in theart. (See, e.g., Peruski and Peruski, The Internet and the New Biology:Tools for Genomic and Molecular Research (ASM Press, Inc. 1997); Wu etal. (eds.), “Information Superhighway and Computer Databases of NucleicAcids and Proteins,” in Methods in Gene Biotechnology 123-151 (CRCPress, Inc. 1997); Bishop (ed.), Guide to Human Genome Computing (2nded., Academic Press, Inc. 1998).) Two amino acid sequences areconsidered to have “substantial sequence identity” if the two sequenceshave at least 80%, at least 90%, or at least 95% sequence identityrelative to each other.

Percent sequence identity is determined by conventional methods. See,e.g., Altschul et al., Bull. Math. Bio. 48:603, 1986, and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1992. For example, twoamino acid sequences can be aligned to optimize the alignment scoresusing a gap opening penalty of 10, a gap extension penalty of 1, and the“BLOSUM62” scoring matrix of Henikoff and Henikoff, supra, as shown inTable 1 (amino acids are indicated by the standard one-letter codes).The percent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

TABLE 1 BLOSUM62 Scoring Matrix A R N D C Q E G H I L K M F P S T W Y VA 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 02 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1−3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1−3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3−1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0−1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2−1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2−3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −33 1 −2 1 −1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and a second amino acid sequence. TheFASTA algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990.Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., residues 19-267 or 25-267 ofSEQ ID NO:2) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63, 1990.

When such a value is expressed as “about” X or “approximately” X, thestated value of X will be understood to be accurate to ±10%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates cholesterol efflux in BHK cell cultures ApoA-1molecules and recombinant fusions thereof. ApoA-1-Fc fusion proteincontaining a 26 amino acid linker between ApoA-1 and the Fc region(ApoA-1(26)Fc) demonstrated increased cholesterol efflux as compared toeither an ApoA-1-Fc fusion protein with a two amino acid linker(ApoA-1(2)Fc (Theripion)) or an ApoA-1-Fc fusion protein without alinker (ApoA-1(0)Fc (Sino Biol)) and had activity similar to wild-typehuman ApoA-1 (Control ApoA-1). ApoA-1 molecules were incubated for 2hours with H3-cholesterol labeled BHK cells induced for ABCA1expression. The Fc proteins were predicted to be dimers; however, theconcentrations shown were calculated and normalized based on the mass ofApoA-1 per molecule.

FIGS. 2A and 2B show schematic diagrams of certain embodiments of fusionproteins in accordance with the present disclosure, including componentfunctional domains. FIG. 2A depicts a schematic representation of ahuman ApoA-1 joined at the carboxyl terminus, via a linker, to a humanIgG Fc region (also referred to herein as a “THER fusion protein” or“THER molecule”). FIG. 2B depicts a schematic representation of a THERfusion protein further joined at the carboxyl terminus, via a linker, toan enzyme region (these fusions are also referred to herein a“Bifunctional Enzyme Lipid Transport” or “BELT” molecule; a BELTmolecule may also be generally referred to herein as a THER fusionprotein or molecule). The linker sequence and the domain present at thecarboxyl terminus of the fusion protein varies depending on theconstruct.

FIG. 3 shows a Western blot of culture supernatants (serum free) fromtransiently transfected 293T cells expressing five different THERmolecules. Transfections and Western blot analysis were performed asdescribed in Example 3, infra. From left to right: MOCK—mocktransfection negative control; CD40IgG—CD40IgG DNA transfection positivecontrol; THER0—ApoA1-IgG fusion protein with a linker of two aminoacids; THER2—ApoA1-IgG fusion protein with a linker of 16 amino acids;THER4—ApoA1-IgG fusion protein with a linker of 26 amino acids;THER6—ApoA1-IgG fusion protein with a linker of 36 amino acids;THER4RNA—ApoA1-IgG fusion protein with a linker of 36 amino acids,further linked via a second 18 amino acid linker to human RNase1.

FIGS. 4A-4E show columnar graphs summarizing the initial screening ofstable CHO clones expressing THER0 (FIG. 4A), THER2 (FIG. 4B), THER4(FIG. 4C), THER6 (FIG. 4D), and THER4RNA2 (FIG. 4E) ApoA-1 fusionproteins and relative expression levels of the fusion proteins from 96well culture supernatants (see Example 4, infra).

FIGS. 5A-5C show results from analysis of a subset of THER clones thatexpressed higher levels of fusion protein, assessing their cell growthpattern (FIG. 5A), relative cell viability (FIG. 5B), and expression offusion protein (FIG. 5C) after six and ten days of culture (see Example4, infra).

FIGS. 6A and 6B show nonreducing (FIG. 6A) and reducing (FIG. 6B)SDS-PAGE analysis of THER fusion proteins purified from CHO clone spentculture supernatants (see Example 4, infra).

FIG. 7 shows Native PAGE gel analysis of the purified THER fusionproteins. Samples were prepared and BLUE Native PAGE gels were run andstained as described in Example 4, infra.

FIG. 8 shows a graph summarizing the relative binding of the differentfusion proteins in a sandwich ELISA, using an anti-IgG capture of fusionproteins and detection step with an HRP-conjugated anti-ApoA-1 antibody(see Example 5, infra).

FIG. 9 shows results from a kinetic enzyme assay measuring the RNaseactivity present in samples of serial dilutions of purifiedApoA1-IgG-RNase bispecific fusion protein (THER4RNA2). An RNASEALERT™assay (IDT, Coralville, Iowa) was performed as described in Example 6,infra, using RNase A (“RNase”) as a positive control andApoA-1-lnk26-hIgG (“THER4”) as a negative control. Each box displays therelative fluorescence units observed as a function of time during thecourse of a 45 minute assay, with a fixed concentration of anon-fluorescent RNA substrate that generates a fluorescent signal upondigestion of the RNA.

FIG. 10 shows a subset of the data shown in FIG. 9, comparing the RNaseenzyme activity at the 4 pmol/μl protein dilution.

FIG. 11 shows the results of a BODIPY-cholesterol efflux assay usingpurified fusion proteins and differentiated human monocytic cell line,THP-1. Assays were performed in a 96 well plate format as described inExample 7, infra, and data are displayed as the mean efflux observedfrom 5 replicates, with baseline efflux (media alone) subtracted fromall samples.

FIG. 12 shows the results of a cholesterol efflux assay using the mousemonocyte-macrophage cell line J774 A.1 (ATCC, Manassas, Va.). Bothbaseline and cAMP-stimulated efflux were assessed as described inExample 7, infra.

FIG. 13 shows a Western blot of culture supernatants (serum free) fromtransiently transfected 293T cells expressing three THER4PON1 sequencevariants (192Q, 192R, 192K) or THER4. Supernatant from mock transfectedcells (Mock transfection) was used as a negative control. Transfectionsand Western blot analysis were performed as described in Example 10,infra.

FIG. 14 shows results of an assay measuring the PON1 organophosphataseactivity in samples of dialyzed culture supernatants from transfectedclones of three THER4PON1 sequence variants (T4P1-192Q, T4P1-192K,T4P1192R). PON1 enzyme (EnzCheck organophosphatase) and untransfectedCHO supernatant were used as positive and negative controls,respectively. Organophosphatase enzyme assays were performed asdescribed in Example 11, infra.

FIG. 15 shows results of an assay measuring the PON1 organophosphataseactivity of THER4PON1 sequence variants (T4P1-192Q, T4P1-192R,T4P1-192K) purified from spent CHO culture supernatants. PON1 enzyme(OPase) and THER4 fusion protein were used as positive and negativecontrols, respectively. Control paraoxonse enzyme was used at twodifferent dilutions (50 mU and 20 mU). Purification of fusion proteinwas performed as described in Example 10, infra, and organophosphataseenzyme assays were performed as described in Example 11, infra.

FIG. 16 shows results of an assay measuring arylesterase activity ofTHER4PON1 sequence variants (T4P1-192Q, T4P1-192R, T4P1-192K) purifiedfrom spent CHO culture supernatants. THER4 fusion protein was used as anegative control. Purification of fusion protein was performed asdescribed in Example 10, infra, and arylesterase activity was measuredas described in Example 11, infra.

FIG. 17 shows results of PK analysis of purified ApoA1-lnk-hIgG-PON1fusion protein after injection into wild-type mice. See Example 12,infra. The data shown summarize plasma concentrations (ng/ml) ofTHER4PON1 192R at different time points following injection.

DESCRIPTION OF THE INVENTION I. Overview

The present invention provides compositions and methods relating tofusion polypeptides comprising a first polypeptide segment havingcholesterol efflux activity and which is either an ApoA1 polypeptide orfunctional variant or fragment thereof or, alternatively, an ApoA-1mimetic. In some aspects, the fusion polypeptide further includes adimerizing domain with a peptide linker between the amino-terminal endof the dimerizing domain and the carboxyl-terminal end of the ApoA-1polypeptide, variant, fragment, or mimetic, thereby allowing the fusionpolypeptide to form stable dimers. In other, non-mutually exclusiveaspects, the fusion polypeptides are bispecific constructs furthercomprising a second polypeptide segment carboxyl-terminal to the ApoA-1polypeptide, variant, fragment, or mimetic and which confers a secondbiological activity. Exemplary second polypeptides include RNases,paraoxonases, platelet-activating factor acetylhydrolases (PAF-AHs),cholesterol ester transfer proteins (CETPs), lecithin-cholesterolacyltransferases (LCATs), polypeptides that specifically bind toproprotein convertase subtilisin/kexin type 9 (PCSK9) and inhibit PCSK9activity, and polypeptides that specifically bind to amyloid beta, anyof which may be a naturally occurring protein or a functional variant orfragment thereof.

The fusion molecules of the present invention can be used, for example,to increase reverse cholesterol transport in a subject and providetherapeutic benefit in the treatment of various diseases. ApoA-1, themajor protein of HDL, has already shown beneficial activity in clinicaltrials in patients with acute coronary syndrome. The ApoA-1 fusionmolecules of the present invention can be used to treat coronary heartdisease, acute coronary syndrome, and other cardiovascular diseasescharacterized by atherosclerosis such as, e.g., stroke. Fusion moleculesof the present invention are also useful, for example, for the treatmentof autoimmune diseases (e.g., rheumatoid arthritis, systemic lupuserythematosus), inflammatory diseases, type 2 diabetes, obesity, andneurodegenerative diseases (e.g., Alzheimer's disease). In someembodiments, fusion proteins of the present invention are used toreplace defective ApoA-1 such as, for example, in the treatment of type1 diabetes and dementia. In certain variations, fusion proteins asdisclosed herein are used to treat multiple sclerosis (MS). ApoA-1levels have been shown to be low in patients with MS, and ApoA-1deficient mice have been shown to exhibit more neurodegeneration andworse disease in experimental allergic encephalomyelitis (EAE), a modelfor MS, than wild-type animals. See Meyers et al., J. Neuroimmunol. 277:176-185, 2014. Data further suggests a positive neuroprotective effectof ApoA-1 on the central nervous system. See Gardner et al., Frontiersin Pharmacology: 20 Nov. 2015 doi: 10.3389/fphar.2015.00278.

Several studies support the use of ApoA-1 therapy for autoimmunedisease. For example, patients with systemic lupus erythematosus (SLE)have low HDL-cholesterol levels and the HDL that is present is oftendamaged by myeloperoxidase-mediated methionine oxidation and tyrosinechlorination of ApoA-1, resulting in loss of ABCA1-dependent cholesterolefflux activity. See Shao et al., J. Biol. Chem. 281:9001-4, 2006;Hewing et al., Arterioscler. Thromb. Vasc. Biol. 34:779-89, 2014. Thispromotes loss of anti-inflammatory properties and generation ofproinflammatory HDL seen in patients with SLE. See Skaggs et al., Clin.Immunol. 137:147-156, 2010; McMahon et al., Athritis Rheum.60:2428-2437, 2009. Autoantibodies to ApoA-1 are present in manypatients with SLE, and SLE-disease activity assessed by SLEDAI and SLEdisease related organ damage assessed by SLICC/ACR damage index arepositively correlated with anti-ApoA-1 antibodies. See Batukla et al.,Ann. NY Acad. Sci. 1108:137-146, 2007; Ahmed et al., EXCLI Journal12:719-732, 2013. Further, increased ApoA-1 concentration attenuatedautoimmunity and glomerulonephritis in lupus prone SLE 1,2,3 mice. SeeBlack et al., J. Immunol. 195:4685-4698, 2015.

Cholesterol efflux capacity of HDL is also impaired in rheumatoidarthritis patients with high disease activity and is correlated withsystemic inflammation and loss of HDL antioxidant activity. SeeCharles-Schoeman et al., Arthritis Rheum. 60:2870-2879, 2009;Charles-Schoeman et al., Ann. Rheum. Dis. 71:1157-1162, 2012. Treatmentof arthritis in the Lewis rat by ApoA-1 and reconstituted HDL reducedacute and chronic joint inflammation, and decreased macrophage TLR2expression and activation. See Wu et al., Arterioscler. Thromb. Basc.Biol. 34:543-551, 2014. Therapy of collagen-induced arthritis in ratswith ApoA-1 mimetic peptide D-4F in combination with pravastatinsignificantly reduced disease activity. See Charles-Schoeman et al.,Clin. Immunol. 127:234-244, 2008.

Fusion molecules of the present invention may also be used in thetreatment of infectious disease. During infection and endotoxemia,significant alterations in lipid metabolism and lipoprotein compositionoccur, including a reduction in ApoA-1 and changes in HDL compositionand size. HDL can bind and neutralize Gram-negative LPS andGram-positive lipoteichoic acid, promoting clearance of theseinflammatory products. Pharmacological studies support the benefit ofrecombinant ApoA-1 during bacterial infection. See, e.g., Pirillo etal., Handb Exp Pharmacol. 224:483-508, 2015.

Fusion molecules of the present invention may be used to treat sepsis.Low levels of HDL have been associated with higher mortality in sepsispatients. See Morin et al., Front. Pharmacol. 6:244, 2015; Monigari etal., International Journal of Scientific and Research Publications, Vol.5, Issue 7, 2015; Tanaka et al., Ann. Intensive Care 7:60, 2017. Inaddition, low levels of PON1 have been reported in sepsis patients andare associated with higher mortality. See Bojic et al., Disease Markers,Vol. 2014, Article ID 427378, 2014; Inal et al., Balkan Med. J.32:183-188, 2015. Supplementation with molecules of the presentinvention, including, for example, bifunctional ApoA-1 fusion moleculesthat contain a paraoxonase (e.g., PON1), can promote anti-inflammatoryprocesses in the recipient that improve clinical outcome.

Bifunctional ApoA-1 fusion molecules of the present invention thatcontain a paraoxonase (e.g., PON1) are particularly useful for therapyof patients infected with Pseudomonas aeruginosa, a gram negativebacterium. This is particularly important for immunocompromisedpatients, where infections with P. aeruginosa are common. P. aeruginosasecrete virulence factors and form biofilm in response to smallsignaling molecules called acyl-homoserine lactones in aconcentration-dependent process called quorum sensing (QS). Paraoxonase1 degrades acyl-homoserine lactones and was shown to protect fromlethality from P. aeruginosa in a transgenic in vivo model in Drosophilamelanogaster where there are no endogenous PON homologs. See Estin etal., Adv. Exp. Med. Biol. 660:183-193, 2010.

Fusion molecules of the present invention may also be used in thetreatment of inflammatory disease. For example, ApoA-1 fusionpolypeptides and dimeric proteins as described herein may alter thephenotype of neutrophils, macrophages, and/or antigen-presenting cellsto reduce proinflammatory responses. Molecules of the present inventioncause efflux of cholesterol from cell membranes, mediated by transportermolecules such as, e.g., ABCA1. Efflux of cholesterol fromantigen-presenting cells, including macrophages and dendritic cells, caninhibit proinflammatory responses mediated by these cells, resulting inreduced production of inflammatory cytokines. Studies support thebenefit of ApoA-1 in mediating anti-inflammatory effects. For example,treatment with ApoA-1 was shown to inhibit the proinflammatory signalingin macrophages after stimulation of CD40 by altering the composition oflipid rafts. See Yin et al., J. Atherosclerosis and Thrombosis19:923-36, 2012. ApoA-1 was also shown to cause a decrease in TRAF-6recruitment to lipid rafts, and a decrease in activation of NF-kB. Seeid. Another study showed that treatment of human monocytes andmacrophages with ApoA-1 or ApoA-1 mimetic 4F altered their response toLPS, resulting in decreased production of inflammatory cytokines MCP-1,MIP-1, RANTES, IL-6, and TNFα, but increased the production of IL-10.See Smythies et al., Am. J. Physiol. Cell Physiol. 298:C1538-48, 2010.doi:1152/ajpce11.00467.2009. Another study showed that treatment withApoA-1 significantly decreased LPS-induced MCP-1 release from THP-1cells, and inhibited expression of CD11b and VCAM-1. See Wang et al.,Cytokine 49:194-2000, 2010. Thus ApoA-1 inhibits activation and adhesionof human monocytes and macrophages, and induces profound functionalchanges due to a differentiation to an anti-inflammatory phenotype.

Inflammatory lung diseases are among inflammatory diseases that may betreated with ApoA-1 fusion molecules as described herein. Serum ApoA-1was found to be positively correlated with FEV1 in patients withcombined atopy and asthma, but not in atopic and nonatopic subjectswithout asthma. See Barochia et al., Am. J. Respir. Crit. Care Med.191:990-1000, 2015. In another study, patients with idiopathic pulmonaryfibrosis had low levels of ApoA-1 in bronchiolar lavage fluid comparedto controls (P<0.01). See Kim et al., Am. J. Respir. Crit. Care Med.182:633-642, 2010. Further, intranasal treatment with ApoA-1 in micetreated with bleomycin was very effective in reducing the number ofinflammatory cells and collagen deposition in the lungs. See id.

Obesity is another inflammatory disease amenable to treatment withApoA-1 fusion molecules in accordance with the present invention.Evidence supports the use of ApoA-1 and HDL to combat obesity. See,e.g., Mineo et al., Circ. Res. 111:1079-1090, 2012. For example,overexpression of ApoA-1 or administration of the ApoA-1 mimetic peptideD-4F has been shown to decrease white adipose mass and insulinresistance and increase energy expenditure in mice fed a high-fat diet.Further, in ob/ob mice, the ApoA-1 mimetic L-4F was shown to loweradiposity and inflammation and improve glucose tolerance. Id.

Yet another disorder that may be treated with ApoA-1 fusion molecules inaccordance with the present invention is nephrotic syndrome (NS), whichis associated with a higher risk for cardiovascular disease in patients.Urinary wastage of filterable HDL (i.e., HDL3) and lipid-poor apo A1 isa common feature of patients with nephrotic syndrome. This is typicallydue to decreased reuptake of these molecules via cubulin/megalinreceptors in the renal proximal tubule. See Barth et al., TrendsCardiovasc. Med. 11:26-31, 2001. ApoA-1 fusion molecules comprising anFc region as described herein would bypass the need for reuptake in thisusual manner, since the molecules are being recycled via FcRn due to thepresence of the Fc domain.

Fusion molecules as described herein may also be used for therapy ofpatients with cancer. It is expected that ApoA-1 fusion polypeptides anddimeric proteins of the present invention, while reducingproinflammatory responses, enhance activation and tumor infiltration ofCD8⁺ T-cells. Studies support the efficacy of ApoA-1 therapy in animalmodels of cancer and have shown that ApoA-1 therapy can cause a specificincrease in CD8⁺ T cells in tumors. See, e.g., Zamanian-Daryoush et al.,J. Biol. Chem. 288:21237-21252, 2013. In some aspects, ApoA-1 fusionmolecules of the present invention are useful in combination with one ormore other anti-cancer therapies such as, for example, an anti-cancerimmunotherapy.

In certain aspects, the present invention provides a way to stabilize anactive ApoA-1 dimer while also controlling the maturation from pre-betaparticles to discoid particles and spherical particles by providing aflexible linker between a dimerizing domain (e.g., an Fc domain) and theC-terminus of the ApoA-1 polypeptide, or functional variant, fragment,or mimetic thereof. A previous ApoA-1-Ig molecule not containing alinker exhibits low activity in cholesterol efflux assays compared towild-type ApoA1. In contrast, dimerizing fusion polypeptides of thepresent invention retain ApoA-1 activity in cholesterol efflux assaysand also allow for further improvements such as, e.g., fusion of anRNase (e.g., RNase 1) or other polypeptide segments C-terminal to thedimerizing domain. In certain preferred embodiments, the use of an Fcregion as the dimerizing domain also allows for increased half-life ofthe dimer.

While not intending to be bound by theory, it is believed that thelength of the linker controls the ability of the stable ApoA-1 dimer toexpand as it takes up cholesterol. The invention provides ApoA-1 fusionmolecules containing flexible linkers between the C-terminus of anApoA-1 polypeptide, or variant, fragment, or mimetic thereof, and theN-terminus of a dimerizing domain such as, e.g., an Fc domain. Linkersare of sufficient length to allow ApoA-1, or the functional variant,fragment, or mimetic thereof, to mediate cholesterol efflux from cells,an initial and critical step in Reverse Cholesterol Transport (RCT).Linkers are typically between 2 and 60 amino acids in length. It isbelieved that ApoA-1 fusion molecules with alternative linker lengthshave distinct functional properties by controlling the maturation of theHDL particle by constraining the C-terminus of ApoA-1. HDL discoidparticles of intermediate size may have improved atheroprotectiveproperties, and may have improved CNS transport properties. Themolecules of this invention may change the progress of HDL maturation atthese intermediate discoid stages, thereby improving efficacy of thefusion proteins of the invention relative to wild type ApoA-1 proteins.The molecules of this invention are likely to affect the structure andcomposition of spherical HDL particles which are composed of trimericApoA-1 particles (see Silva et al., Natl. Acad. Sci. USA105:12176-12181, 2008). It is likely that molecules of this inventionwill interact with natural ApoA-1 in the formation of larger sphericalHDL particles.

In certain embodiments, the dimerizing domain is a immunoglobulin Fcregion. ApoA-1-Fc fusion molecules of the present invention extendApoA-1 half-life while retaining ApoA-1 reverse cholesterol efflux andeliminating the requirement for extensive lipid formulation. Inaddition, the presence of the Fc region allows purification usingimmobilized Protein A according to standard practices in and antibodyand Fc fusion protein manufacturing.

Structural studies of ApoA-1 (see, e.g., Gogonea, Frontiers Pharmacol.6:318, 2016) show that ApoA-1 assumes multiple conformations as itmatures from lipid-free monomer to higher order forms. Recent dataderived from small angle neutron scattering (SANS) show low resolutionstructures of ApoA-1 dimers in an open configuration around a lipidcore, called the super double helix (DSH) model. Other structures fromSANS studies show ApoA-1 in different open configurations depending onthe composition of the lipid core; in these structures, the C-terminusof the ApoA-1 monomers are in different positions relative to eachother. Similarly, spherical ApoA-1 particles that incorporate a thirdApoA-1 monomer show the C-terminus of each monomer in a differentposition compared to the positions in dimeric discoid ApoA-1. See, e.g.,Gogonea, supra. The flexible linkers of the present disclosure are ofsufficient length to allow ApoA-1 to assume these positions withoutconformational constraint.

In certain embodiments, ApoA-1-[linker]-[dimerizing domain] molecules ofthe present invention include an additional polypeptide segment fusedcarboxyl-terminal to the dimerizing domain. Such variations allow forthe creation of bispecific molecules with ApoA-1 functional activity anda second biological activity.

In some aspects of the present invention, bispecific fusion moleculesare provided comprising a (i) first polypeptide segment with reversecholesterol transport activity and which is either an ApoA1 polypeptideor functional variant or fragment thereof or, alternatively, an ApoA1mimetic and (ii) a second polypeptide segment carboxyl-terminal to thefirst polypeptide segment, wherein the second polypeptide segment isselected from an RNase, a paraoxonase, a platelet-activating factoracetylhydrolase (PAF-AH), a cholesterol ester transfer protein (CETP), alecithin-cholesterol acyltransferase (LCAT), a polypeptide thatspecifically binds to proprotein convertase subtilisin/kexin type 9(PCSK9) and inhibits PCSK9 activity, and a polypeptide that specificallybinds to amyloid beta. Such second polypeptides may be a naturallyoccurring protein or a functional variant or fragment thereof. In someembodiments, a linker and dimerizing domain is included between thefirst and second polypeptides as summarized above. In alternativeembodiments, the fusion polypeptide lacks a dimerizing domain.

In some embodiments of the present ApoA-1 fusion molecules that lack anFc region, the fusion molecule may be conjugated to PEG to provideextended half-life. Such variations may include bispecific molecules asdescribed herein, such as, e.g., fusion molecules comprising an RNase, aparaoxonase, a platelet-activating factor acetylhydrolase (PAF-AH), acholesterol ester transfer protein (CETP), a lecithin-cholesterolacyltransferase (LCAT), a polypeptide that specifically binds toproprotein convertase subtilisin/kexin type 9 (PCSK9) and inhibits PCSK9activity, or a polypeptide that specifically binds to amyloid beta.

In some embodiments of a bispecific molecule as summarized above, thesecond polypeptide segment is an RNase. A preferred RNase is human RNase1 or a functional variant or fragment thereof. In particular variations,the RNase retains its sensitivity to inhibition by cytoplasmic inhibitorand has very low toxicity to cells, but is highly activeextracellularly. RNase has anti-inflammatory properties by digestion ofinflammatory extracellular RNA and provides additional therapeuticbenefit for treatment of various diseases, including cardiovasculardiseases (e.g., coronary artery disease, stroke), autoimmune diseases,inflammatory diseases, type 2 diabetes, infectious disease, andneurodegenerative diseases (e.g., Alzheimer' disease).

For example, a bispecific ApoA-1 fusion molecule comprising an RNasesegment as described herein may be used, e.g., for treatment of aninflammatory disease such as, for example, an inflammatory lung disease.One study has shown that TLR3, an RNA sensor, has a major role in thedevelopment of ARDS-like pathology in the absence of a viral pathogen.See Murray et al., Am. J. Respir. Crit. Care Med. 178:1227-1237, 2008.Oxygen therapy is a major therapeutic intervention in ARDS, butcontributes to further lung damage and susceptibility to viralinfection. Oxygen therapy was a major stimulus for increased TLR3expression and activation in cultured human epithelial cells, andabsence or blockade of TLR3 protected mice from lung injury andinflammation after exposure to hyperoxic conditions. See Murray et al.,supra. Another study has shown that TLR3 activation by extracellular RNAoccurs in response to acute hypoxia, and that therapy in mice withRNaseA diminished lung inflammation after acute hypoxia. See Biswas etal., Eur. J. Immunol. 45: 3158-3173, 2015. A bispecific ApoA-1 fusionmolecule comprising an RNase segment as described herein may also beused, e.g., for treatment of an autoimmune disease such as, for example,systemic lupus erythematosus (SLE). Studies show, for example, a role ofRNA immune complexes and RNA receptors, including TLR7, in SLE diseasepathogenesis, as well as a protective effect of RNase overexpression inmouse models of SLE. See, e.g., Sun et al., J. Immunol. 190:2536-2543,2013.

In other embodiments of a bispecific molecule as summarized above, thesecond polypeptide segment is a paraoxonase. A preferred paraoxonase ishuman paraoxonase 1 (PON1) or a functional variant or fragment thereof.Paraoxonase has multiple activities including organophosphatase,phosphotriesterase, arylesterase, and thiolactonase. Theorganophosphatase activity confers protection against toxicorganophosphates including insecticides such as paraoxon. Paraoxonasebispecific fusion molecules provide additional therapeutic benefit forthe treatment of diseases amenable to ApoA-1-mediated therapy,including, for example, through its atheroprotective, antioxidant,anti-inflammatory, and/or neuroprotective properties. In somealternative embodiments, PON1 may attached to an ApoA-1 fusion moleculeof the present invention through its natural, high affinity binding toApoA-1, which binding is mediated by Tyr71 of PON1 (see Huang et al., J.Clin. Invest. 123:3815-3828, 2013). Incubating an ApoA-1 fusion moleculewith recombinant or natural PON1 prior to administration will besufficient to “load” PON1 onto the ApoA-1 fusion molecule.

A bispecific ApoA-1 fusion molecule comprising a paraoxonase segment asdescribed herein may be used, e.g., for treatment of an autoimmunedisease or an inflammatory disease. For example, studies support use ofa paraoxonase for treatment of autoimmune disease such as systemic lupuserythematosus (SLE). The autoantibody titer in many patients withsystemic lupus erythematosus (SLE) is correlated with loss of activityof PON1 (see Batukla et al., Ann. NY Acad. Sci. 1108:137-146, 2007), andSLE-disease activity assessed by SLEDAI and SLE disease related organdamage assessed by SLICC/ACR damage index are negatively correlated withPON1 activity (see Ahmed et al., EXCLI Journal 12:719-732, 2013). PON1activity is significantly reduced in patients with SLE, and is a riskfactor for atherosclerosis. See Kiss et al., Ann. NY Acad. Sci.108:83-91, 2007. In addition, other studies support use of a paraoxonasefor treatment of inflammatory disease such as inflammatory lungdiseases. One study showed that patients with late lung diseases longafter exposure to sulfur mustard gas (SM), including asthma, chronicobstructive pulmonary disease (COPD) and bronchiectasis, havesignificantly reduced levels of PON1 in bronchiolar lavage fluid(p<0.0001). See Golmanesh et al., Immunopharmacol. Immunotoxical.35:419-425, 2013. Another study showed that Iranian veterans exposed toSM twenty years ago still have significantly low serum levels of PON1activity, and low PON1 was correlated with lung disease severity. SeeTaravati et al., Immunopharmacol. Immunotoxicol. 34:706-713, 2012.

Bispecific ApoA-1 fusion molecules comprising either an RNase segment ora paraoxonase segment as described herein may also be used, e.g., fortreatment of a neurological disease. Such bispecific molecules aretransported to the brain where they deliver a protective paraoxonase orRNase enzyme. For example, PON1 is protective in the brain because ofits antioxidant properties, and RNase is protective by digestingextracellular RNA that promotes inflammation via stimulation of TLR7 andother RNA receptors. Exemplary neurological diseases amenable totreatment using an ApoA-1/paraoxonase or ApoA1/RNase bispecific moleculeof the present invention include multiple sclerosis, Parkinson'sdisease, and Alzheimer's disease.

Attachment of myeloperoxidase (MPO) inhibitors to ApoA-1 fusionmolecules of the present invention may be particularly desirable as away to protect ApoA-1 from inactivation due to oxidation mediated byMPO, and can also similarly protect paraoxonase from MPO-mediatedoxidation and inactivation in the context of a bispecific fusionpolypeptide comprising a paraoxonase such as PON1.Myeloperoxidase-mediated oxidation of ApoA-1 promotes crosslinking ofApoA-1, and may be implicated in the mechanism that leads to amyloiddeposition in atherosclerotic plaques in vivo. See Chan et al., J. Biol.Chem. 290: 10958-71, 2015. For a review of MPO inhibitors, see Malle etal., Br J Pharmacol. 152: 838-854, 2007. The attachment of a MPOinhibitor to a molecule of the present invention can also localize theMPO inhibition to selectively protect ApoA-1 from oxidation whilepreserving MPO activity important in anti-microbial activity.

In other embodiments of a bispecific molecule as summarized above, thesecond polypeptide segment is selected from a cholesterol ester transferprotein (CETP), and a lecithin-cholesterol acyltransferase (LCAT). CETPis involved in one of the major mechanisms by which HDL particles candeliver cholesterol to the liver during the process of reversecholesterol transport (RCT), specifically, through unloading andtransferring of cholesterol to LDL, which then transports cholesterolback to the liver via LDL receptors. This process of unloading requiresCETP. By improving the initial part of the RCT pathway through thedelivery of improved ApoA-1 molecules such as provided herein, thenadding other RCT components can provide an attractive and potentiallysynergistic therapeutic approach. Providing more exogenous CETP in theform of a bispecific fusion molecule containing ApoA-1 can enhance CETPactivity and overall reverse cholesterol transport.

Bispecific fusions containing LCAT can provide an alternative means ofenhancing endogenous CETP. Lecithin-cholesterol acyltransferase (LCAT)is an enzyme that is associated with HDL and converts free cholesterolto cholesteryl esters, which is then sequestered into the HDL particleand allows for its spherical shape formation. A human recombinant LCATgiven to mice lacking LCAT significantly improved HDL-C levels, and whengiven to human ApoA-1 transgenic mice, the increase in HDL-C waseight-fold, suggesting synergy. See Rousset et al., J Pharmacol ExpTher. 335:140-8, 2010. A recombinant human LCAT fusion to Fc has beenreported (see Spahr et al., Protein Sci. 22:1739-53, 2013), and abispecific molecule containing both ApoA-1 and LCAT may also improve RCTmore efficiently than a mono-specific protein of either alone.

In other embodiments of a bispecific molecule as summarized above, thesecond polypeptide segment is a polypeptide that specifically binds toproprotein convertase subtilisin/kexin type 9 (PCSK9) and inhibits PCSK9activity. In some variations, a PCSK9-binding polypeptide inhibits PCSK9activity by inhibiting its binding to the LDL receptor. In somevariations, a PCSK9-binding polypeptide is a PCSK9-specific single chainantibody such as, for example, a PCSK9-specific scFv. Anti-PCSK9antibodies that inhibit PCSK9 activity are generally known in the art(see, e.g., International PCT Publication Nos. WO 2008/057459, WO2010/077854, and WO 2012/109530; US Patent Application Publication No.2011/0142849), and anti-PCSK9 monoclonal antibodies have been approvedby FDA for treatment of hypercholesterolemia. A bispecific molecule thatmediates cholesterol efflux and inhibits PCSK9 is expected to be apotent therapy for vascular disease because it would reduce inflammationthrough multiple pathways. Cholesterol efflux from macrophages andneutrophils reduces inflammatory cytokines and myeloperoxidaseproduction and provides the beneficial effects of HDL, while inhibitionof PCSK9 increases expression of the LDL receptor, thus reducinginflammatory LDL.

In other embodiments of a bispecific molecule as summarized above, thesecond polypeptide segment is a polypeptide that specifically binds toamyloid beta (Aβ). In a specific variation, the second polypeptide is aAβ-specific single chain antibody such as, for example, an Aβ-specificscFv. A scFv specific for amyloid beta peptide is described, forexample, by Cattepoel et al., PLoS One 6:e18296, 2011. In suchembodiments, the Aβ-binding polypeptide is typically fused C-terminal toApoA-1, or C-terminal to the dimerizing domain, if present. Thisbispecific fusion molecule has improved properties for therapy ofpatients with Alzheimer's disease.

II. Fusion Polypeptides and Dimeric Proteins

Accordingly, in one aspect, the present invention provides a fusionpolypeptide comprising, from an amino-terminal position to acarboxyl-terminal position, ApoA1-L1-D, where ApoA1 is a firstpolypeptide segment having cholesterol efflux activity and which isselected from (i) a naturally occurring ApoA-1 polypeptide or afunctional variant or fragment thereof and (ii) an ApoA-1 mimetic; L1 isa first polypeptide linker; and D is a dimerizing domain. In someembodiments, the fusion polypeptide further includes a secondpolypeptide segment located carboxyl-terminal to the dimerizing domain.In particular variations, the second polypeptide segment is (a) anaturally occurring RNase, paraoxonase, platelet-activating factoracetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), orlecithin-cholesterol acyltransferase (LCAT); (b) a functional variant orfragment of any of the naturally occurring proteins specified in (a); or(c) a polypeptide that specifically binds to amyloid beta (Aβ) such as,e.g., an Aβ-specific scFv. Such a fusion polypeptide comprising a secondpolypeptide segment may be represented by the formula ApoA1-L1-D-L2-P(from an amino-terminal position to a carboxyl-terminal position), whereApoA1, L1, and D are each as previously defined, where L2 is a secondpolypeptide linker and is optionally present, and where P is the secondpolypeptide segment.

In another aspect, the present invention provides a fusion polypeptidecomprising a first polypeptide segment having cholesterol effluxactivity and which is selected from (i) a naturally occurring ApoA-1polypeptide or a functional variant or fragment thereof and (ii) anApoA-1 mimetic, and a second polypeptide segment locatedcarboxyl-terminal to the first polypeptide segment, where the secondpolypeptide segment is (a) a naturally occurring RNase, paraoxonase,platelet-activating factor acetylhydrolase (PAF-AH), cholesterol estertransfer protein (CETP), or lecithin-cholesterol acyltransferase (LCAT);(b) a functional variant or fragment of any of the naturally occurringproteins specified in (a); (c) a polypeptide that specifically binds toproprotein convertase subtilisin/kexin type 9 (PCSK9) and inhibits PCSK9activity such as, e.g., a PCSK9-specific scFv; or (d) a polypeptide thatspecifically binds to amyloid beta (Aβ) such as, e.g., an Aβ-specificscFv. In some variations, the fusion polypeptide further includes alinker polypeptide located carboxyl-terminal to the first polypeptidesegment and amino-terminal to the second polypeptide segment. In someembodiments, the fusion polypeptide further includes a dimerizingdomain, which can be located, for example, carboxyl-terminal to thefirst polypeptide segment and amino-terminal to the second polypeptidesegment.

Functional variants of a particular naturally occurring proteinspecified above can be readily identified using routine assays forassessing the variant for a relevant biological or biochemical activitycorresponding to the natural protein. For example, in the case ofApoA-1, variants may be assayed for their ability to induce cholesterolefflux using known cholesterol efflux assays such as described herein.See, e.g., Tang et al., J Lipid Res. 47:107-14, 2006. In the case ofRNase such as human RNase 1, variants may be assayed for their abilityto digest single or double-stranded RNA is known assays to assessribonuclease activity. See, e.g., Libonati and Sorrentino, MethodsEnzymol. 341234-248, 2001. Paraoxonase 1 (PON1) variants may be assayedfor phosphotriesterase activity using diethyl p-nitrophenol phosphate(paraoxon) as a substrate, or for arylesterase activity using phenylacetate as a substrate. See, e.g., Graves and Scott, Curr Chem Genomics2:51-61, 2008. In addition to these tests, the EnzChek Paraoxonase AssayKit (E33702: ThermoFisher) is a highly sensitive, homogeneousfluorometric assay (excitation/emission maxima ˜360/450 nm) for theorganophosphatase activity of paraoxonase and is based on the hydrolysisof a proprietary, fluorogenic organophosphate analog. This assay hasbeen used in several published studies of PON1 activity in patient sera.See, e.g., Brian et al., Chemosphere 120:479-485, 2015; Rector et al.,Am J Physiol Endocrinol Metab 293:E500-E506, 2007. Assays to assessrelevant CETP and LCAT activities are also known. For example, assaysfor measuring LCAT and CETP enzyme activity are commercially availableand include, e.g., Cell Biolabs Cat. No. STA-615, Sigma-Aldrich Cat. No.MAK107, and Roar Biomedical Cat. No. RB-LCAT for LCAT, and Abcam Cat.No. ab65383 and Sigma-Aldrich Cat. No. MAK106 for CETP.

In the case of Aβ-binding or PCSK9-binding activity, polypeptides suchas, e.g., single chain antibodies may be assessed for binding activityusing any of various known assays. For example, one assay system employsa commercially available biosensor instrument (BIAcore™ PharmaciaBiosensor, Piscataway, N.J.), wherein a binding protein (e.g.,Aβ-binding candidate, such as an antibody) is immobilized onto thesurface of a sensor chip, and a test sample containing a soluble antigen(e.g., Aβ peptide) is passed through the cell. If the immobilizedprotein has affinity for the antigen, it will bind to the antigen,causing a change in the refractive index of the medium, which isdetected as a change in surface plasmon resonance of the gold film. Thissystem allows the determination of on- and off-rates, from which bindingaffinity can be calculated, and assessment of stoichiometry of binding.Use of this instrument is disclosed, e.g., by Karlsson (J. Immunol.Methods 145:229-240, 1991) and Cunningham and Wells (J. Mol. Biol.234:554-563, 1993). Aβ-binding polypeptides can also be used withinother assay systems known in the art. Such systems include Scatchardanalysis for determination of binding affinity (see Scatchard, Ann. NYAcad. Sci. 51: 660-672, 1949) and calorimetric assays (see Cunningham etal., Science 253:545-548, 1991; Cunningham et al., Science 254:821-825,1991).

Naturally occurring polypeptide segments for use in accordance with thepresent invention (e.g., a naturally occurring ApoA-1 polypeptide,RNase, paraoxonase, or platelet-activating factor acetylhydrolase)includes naturally occurring variants such as, for example, allelicvariants and interspecies homologs consistent with the disclosure.

Functional variants of a particular reference polypeptide (e.g., awild-type human ApoA-1) are generally characterized as having one ormore amino acid substitutions, deletions or additions relative to thereference polypeptide. These changes are preferably of a minor nature,that is conservative amino acid substitutions (see, e.g., Table 2,infra, which lists some exemplary conservative amino acid substitutions)and other substitutions that do not significantly affect the folding oractivity of the protein or polypeptide; small deletions, typically ofone to about 30 amino acids; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide, or a small extension that facilitates purification (an affinitytag), such as a poly-histidine tract, protein A (Nilsson et al., EMBO J.4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3, 1991), glutathioneS transferase (Smith and Johnson, Gene 67:31, 1988), or other antigenicepitope or binding domain. (See generally Ford et al., ProteinExpression and Purification 2:95-107, 1991.) DNAs encoding affinity tagsare available from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.). Conservative substitutions may also be selected fromthe following: 1) Alanine, Glycine; 2) Aspartate, Glutamate; 3)Asparagine, Glutamine; 4) Arginine, Lysine; 5) Isoleucine, Leucine,Methionine, Valine; 6) Phenylalanine, Tyrosine, Tryptophan; 7) Serine,Threonine; and 8) Cysteine, Methionine (see, e.g., Creighton, Proteins(1984)).

TABLE 2 Conservative amino acid substitutions Basic Acidic PolarHydrophobic Aromatic Small Arginine Glutamate Glutamine LeucinePhenylalanine Glycine Lysine Aspartate Asparagine Isoleucine TryptophanAlanine Histidine Valine Tyrosine Serine Methionine Threonine Methionine

Essential amino acids in a naturally occurring polypeptide can beidentified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081-1085, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-4502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.,cholesterol efflux for ApoA-1 variants) to identify amino acid residuesthat are critical to the activity of the molecule. In addition, sites ofrelevant protein interactions can be determined by analysis of crystalstructure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. The identities ofessential amino acids can also be inferred from analysis of homologieswith related proteins (e.g., species orthologs retaining the sameprotein function).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer Science 241:53-57, 1988 or Bowie and SauerProc. Natl. Acad. Sci. USA 86:2152-2156, 1989. Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Another method that can be used isregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Variant nucleotide and polypeptide sequences can also be generatedthrough DNA shuffling. (See, e.g., Stemmer, Nature 370:389, 1994;Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747, 1994; InternationalPublication No. WO 97/20078.) Briefly, variant DNA molecules aregenerated by in vitro homologous recombination by random fragmentationof a parent DNA followed by reassembly using PCR, resulting in randomlyintroduced point mutations. This technique can be modified by using afamily of parent DNA molecules, such as allelic variants or DNAmolecules from different species, to introduce additional variabilityinto the process. Selection or screening for the desired activity,followed by additional iterations of mutagenesis and assay provides forrapid “evolution” of sequences by selecting for desirable mutationswhile simultaneously selecting against detrimental changes.

As previously discussed, a polypeptide fusion in accordance with thepresent invention can include a polypeptide segment corresponding to a“functional fragment” of a particular polypeptide. Routine deletionanalyses of nucleic acid molecules can be performed to obtain functionalfragments of a nucleic acid molecule encoding a given polypeptide. As anillustration, ApoA-1-encoding DNA molecules having the nucleotidesequence of residues 70-816 of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for the ability to inducecholesterol efflux. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a gene encoding a polypeptide can be synthesizedusing the polymerase chain reaction.

Accordingly, using methods such as discussed above, one of ordinaryskill in the art can prepare a variety of polypeptides that (i) aresubstantially identical to a reference polypeptide (e.g., residues19-267 or 25-267 of SEQ ID NO:2 for a human wild-type ApoA-1polypeptide) and (ii) retains the desired functional properties of thereference polypeptide.

Polypeptide segments used within the present invention (e.g.,polypeptide segments corresponding to ApoA-1, RNase, paraoxonase,platelet-activating factor acetylhydrolase, dimerizing domains such as,e.g., Fc fragments) may be obtained from a variety of species. If theprotein is to be used therapeutically in humans, it is preferred thathuman polypeptide sequences be employed. However, non-human sequencescan be used, as can variant sequences. For other uses, including invitro diagnostic uses and veterinary uses, polypeptide sequences fromhumans or non-human animals can be employed, although sequences from thesame species as the patient may be preferred for in vivo veterinary useor for in vitro uses where species specificity of intermolecularreactions is present. Thus, polypeptide segments for use within thepresent invention can be, without limitation, human, non-human primate,rodent, canine, feline, equine, bovine, ovine, porcine, lagomorph, andavian polypeptides, as well as variants thereof.

In certain embodiments, the first polypeptide segment is a humanwild-type ApoA-1 polypeptide or a functional variant or fragmentthereof. For example, in some embodiments, the first polypeptide segmentcomprises an amino acid sequence having at least 80% identity with aminoacid residues 19-267 or 25-267 of SEQ ID NO:2. In more particularembodiments, the first polypeptide segment comprises an amino acidsequence having at least 85%, at least 90%, or at least 95% identitywith amino acid residues 19-267 or 25-267 of SEQ ID NO:2. In yet otherembodiments, the first polypeptide segment comprises an amino acidsequence having at least 96%, at least 97%, at least 98%, at least 99%,or 100% sequence identity with amino acid residues 19-267 or 25-267 ofSEQ ID NO:2. In some embodiments, the first polypeptide segment is afunctional variant of human wild-type ApoA-1 comprising one or moreamino acid modifications that confer resistance to oxidation bymyeloperoxidase (MPO). In specific variations, valine at the amino acidposition corresponding to position 156 of mature human wild-type ApoA-1is replaced by glutamate or lysine, and/or arginine at the amino acidposition corresponding to position 173 of mature human wild-type ApoA-1is replaced by cysteine (also referred to herein, respectively, asV156[E/K] and R173C variants, mutations, or substitutions). Position 156of the mature human wild-type ApoA-1 corresponds to amino acid position180 of SEQ ID NO:2, and position 173 of mature human wild-type ApoA-1corresponds to amino acid position 197 of SEQ ID NO:2. V156K and R173Cmutations have improved activity and half-life in atherosclerotic micecompared to wild-type ApoA-1. See Cho et al., Exp Mol Med 41:417, 2009.In some variations, tyrosine at the amino acid position corresponding toposition 192 of mature human wild-type ApoA-1 is replaced by serine,glutamine, asparagine, histidine, or phenylalanine (also referred toherein as a Y192[S/Q/N/H/F] variant, mutation, or substitution).Position 192 of the mature human wild-type ApoA-1 corresponds to aminoacid position 216 of SEQ ID NO:2. In some variations, at least one ofthe methionine residues at the amino acid positions corresponding topositions 86, 112, and 148 of the mature human wild-type ApoA-1 isreplaced with leucine, isoleucine, or valine (also referred to herein asM86[L/I/V], M112[L/I/V], and M148[L/I/V] variants, mutations, orsubstitutions). Positions 86, 112, and 148 of the mature human wild-typeApoA-1 respectively correspond to amino acid positions 110, 136, and 172of SEQ ID NO:2. In some variations, at least one of the tryptophanresidues at the amino acid positions corresponding to positions 8, 50,72, and 108 of the mature human wild-type ApoA-1 is replaced withphenylalanine (also referred to herein W8F, W50F, W72F, and W108Fvariants, mutations, or substitutions); in some such embodiments, allfour of these tryptophan residues are replaced with phenylalanine (alsoreferred to herein as a 4WF variant or mutation). Positions 8, 50, 72,and 108 of the mature human wild-type ApoA-1 respectively correspond toamino acid positions 32, 74, 96, and 132 of SEQ ID NO:2. In someembodiments, the first polypeptide segment is an ApoA-1 variantcomprising at least one of the V156[E/K] and Y192[S/Q/N/H/F]substitutions, and optionally at least one of the M86[L/I/V],M112[L/I/V], and M148[L/I/V] substitutions; in some such embodiments,the first polypeptide segment is an ApoA-1 variant comprising both ofthe V156[E/K] and Y192[S/Q/N/H/F] substitutions. In more particularvariations, the first polypeptide segment is an ApoA-1 variantcomprising the specific V156[E/K] and/or Y192[S/Q/N/H/F] substitution(s)of any one of variant combinations A1-A17 as shown in Table 3, infra.

TABLE 3 Combinations of ApoA-1 V156[E/K] & Y192[S/Q/N/H/F] VariantsApoA-1 Variant Amino Acid at Position No.:† Combination 156 192 A1 E SA2 E Q A3 E N A4 E H A5 E F A6 E Y (wild-type) A7 K S A8 K Q A9 K N A10K H A11 K F A12 K Y (wild-type) A13 V (wild-type) S A14 V (wild-type) QA15 V (wild-type) N A16 V (wild-type) H A17 V (wild-type) F †Amino acidpositions 156 and 192 are according to the mature human wild-type ApoA-1and respectively correspond to amino acid positions 180 and 216 of SEQID NO: 2.

In other embodiments, the first polypeptide segment is an ApoA-1 mimeticsuch as, for example, the 4F peptide (see Song et al., Int. J. Biol.Sci. 5:637-646, 2009). ApoA-1 mimetics are generally known in the artand are reviewed in Reddy et al., Curr. Opin. Lipidol. 25: 304-308,2014.

In certain embodiments comprising a second polypeptide segmentcarboxyl-terminal to the first polypeptide segment (e.g.,carboxyl-terminal to a dimerizing domain), the second polypeptidesegment is an RNase. In some embodiments, the RNase is a human RNAse 1or a functional variant or fragment thereof. For example, in someembodiments, the second polypeptide segment comprises an amino acidsequence having at least 80% identity with amino acid residues 544-675or 548-675 of SEQ ID NO:4. In more particular embodiments, the secondpolypeptide segment comprises an amino acid sequence having at least85%, at least 90%, or at least 95% identity with amino acid residues544-675 or 548-675 of SEQ ID NO:4. In yet other embodiments, the secondpolypeptide segment comprises an amino acid sequence having at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identitywith amino acid residues 544-675 or 548-675 of SEQ ID NO:4.

In other embodiments comprising a second polypeptide segmentcarboxyl-terminal to the first polypeptide segment (e.g.,carboxyl-terminal to a dimerizing domain), the second polypeptidesegment is a paraoxonase. In some embodiments, the paraoxonase is ahuman paraoxonase 1 (PON1) or a functional variant or fragment thereof.For example, in some embodiments, the second polypeptide segmentcomprises an amino acid sequence having at least 80% identity with aminoacid residues 16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQID NO:42, or amino acid residues 16-355 of SEQ ID NO:44. In moreparticular embodiments, the second polypeptide segment comprises anamino acid sequence having at least 85%, at least 90%, or at least 95%identity with amino acid residues 16-355 of SEQ ID NO:12, amino acidresidues 16-355 of SEQ ID NO:42, or amino acid residues 16-355 of SEQ IDNO:44. In yet other embodiments, the second polypeptide segmentcomprises an amino acid sequence having at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity with amino acidresidues 16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQ IDNO:42, or amino acid residues 16-355 of SEQ ID NO:44. (SEQ ID NO:12 is a192Q form, SEQ ID NO:42 is a 192K variant, and SEQ ID NO:44 is a 192Rvariant of a human paraoxonase molecule, where numbering is based onfull length PON1, including the leader peptide.) In some variationswhere the second polypeptide segment is a variant of a human PON1polypeptide as above, tyrosine at the amino acid position correspondingto position 185 of full-length human wild-type PON1 is replaced byhistidine, glutamine, or serine (also referred to herein as aY185[H/Q/S] variant, mutation, or substitution) and/or phenylalanine atthe amino acid position corresponding to position 293 of full-lengthhuman wild-type PON1 is replaced by histidine, glutamine, or asparagine(also referred to herein as a F293[H/Q/N] variant, mutation, orsubstitution). Positions 185 and 293 of the full-length human wild-typePON1 respectively correspond to amino acid positions 185 and 293 of SEQID NO:12, SEQ ID NO:42, or SEQ ID NO:44. In more particular variationswhere the second polypeptide segment is a variant of a human PON1polypeptide as above, the second polypeptide segment comprises thespecific Y185[H/Q/S] and/or F293[H/Q/N] substitution(s) of any one ofvariant combinations P1-P15 as shown in Table 4, infra. Other PON1 aminoacid variants that may be incorporated into a paraoxonase polypeptidesegment in the context of the present invention are disclosed, e.g., inUS Patent Application Publication Nos. 2014/0079682 and 2012/0213834,which are incorporated by reference herein.

TABLE 4 Combinations of PON1 Y185[H/Q/S] & F293[H/Q/N] Variants PON1Variant Amino Acid at Position No.:† Combination 185 293 P1 H H P2 H QP3 H N P4 H F (wild-type) P5 Q H P6 Q Q P7 Q N P8 Q F (wild-type) P9 S HP10 S Q P11 S N P12 S F (wild-type) P13 Y (wild-type) H P14 Y(wild-type) Q P15 Y (wild-type) N †Amino acid positions 185 and 293 areaccording to the full-length human wild-type PON1 and respectivelycorrespond to amino acid positions 185 and 293 of SEQ ID NO: 12, SEQ IDNO: 42, or SEQ ID NO: 44.

In some embodiments of a fusion polypeptide comprising a paraoxonasecarboxyl-terminal to the first polypeptide segment (e.g.,carboxyl-terminal to a dimerizing domain), where the first polypeptidesegment comprises an amino acid sequence having at least 80%, at least85%, at least 90%, or at least 95% identity with amino acid residues19-267 or 25-267 of SEQ ID NO:2 and the second polypeptide segmentcomprises an amino acid sequence having at least 80% identity, at least85%, at least 90%, or at least 95% identity with amino acid residues16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQ ID NO:42, oramino acid residues 16-355 of SEQ ID NO:44, the first polypeptidesegment is an ApoA-1 variant comprising the specific V156[E/K] and/orY192[S/Q/N/H/F] substitution(s) of any one of variant combinationsA1-A17 as shown in Table 3 herein and the second polypeptide segment isa variant of a human PON1 polypeptide comprising the specificY185[H/Q/S] and/or F293[H/Q/N] substitution(s) of any one of variantcombinations P1-P15 as shown in Table 4 herein. Table 5, infra, showsApoA-1 variant combinations A1-A17 from Table 3 arrayed against PON1variant combinations P1-P15 from Table 4, where each “Aβ[44]”designation represents a specific combination of an ApoA-1V156[E/K]/Y192[S/Q/N/H/F] variant with a PON1 Y185[H/Q/S]/F293[H/Q/N]variant (for example, “Aβ20” represents a combination of ApoA1/PON1variants combining ApoA1 variant combination A3 of Table 3 with PON1variant combination P2 of Table 4, and represents the specificcombination of ApoA1 Y156E and Y192N substitutions together with PON1Y185H and F293Q substitutions). A fusion polypeptide comprising aparaoxonase carboxyl-terminal to the first polypeptide segment andcomprising variants from Tables 3 and 4 herein may be a fusionpolypeptide comprising any one of the specific combinations representedby Aβ1-Aβ255 in Table 5; in some such embodiments, the first polypeptideis otherwise 100% identical to residues 19-267 or 25-267 of SEQ ID NO:2and/or the second polypeptide segment is otherwise 100% identical toresidues 16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQ IDNO:42, or amino acid residues 16-355 of SEQ ID NO:44.

TABLE 5 ApoA-1 V156[E/K]/Y192[S/Q/N/H/F] Variants† in Combination withPON1 Y185[H/Q/S]/F293[H/Q/N] Variants‡ P1 P2 P3 P4 P5 P6 P7 PS P9 P10P11 P12 P13 P14 P15 A1 AP1 AP18 AP35 AP52 AP69 AP86 AP103 AP120 AP137AP154 AP171 AP188 AP205 AP222 AP239 A2 AP2 AP19 AP36 AP53 AP70 AP87AP104 AP121 AP138 AP155 AP172 AP189 AP206 AP223 AP240 A3 AP3 AP20 AP37AP54 AP71 AP88 AP105 AP122 AP139 AP156 AP173 AP190 AP207 AP224 AP241 A4AP4 AP21 AP38 AP55 AP72 AP89 AP106 AP123 AP140 AP157 AP174 AP191 AP208AP225 AP242 A5 AP5 AP22 AP39 AP56 AP73 AP90 AP107 AP124 AP141 AP158AP175 AP192 AP209 AP226 AP243 A6 AP6 AP23 AP40 AP57 AP74 AP91 AP108AP125 AP142 AP159 AP176 AP193 AP210 AP227 AP244 A7 AP7 AP24 AP41 AP58AP75 AP92 AP109 AP126 AP143 AP160 AP177 AP194 AP211 AP228 AP245 A8 AP8AP25 AP42 AP59 AP76 AP93 AP110 AP127 AP144 AP161 AP178 AP195 AP212 AP229AP246 A9 AP9 AP26 AP43 AP60 AP77 AP94 AP111 AP128 AP145 AP162 AP179AP196 AP213 AP230 AP247 A10 AP10 AP27 AP44 AP61 AP78 AP95 AP112 AP129AP146 AP163 AP180 AP197 AP214 AP231 AP248 A11 AP11 AP28 AP45 AP62 AP79AP96 AP113 AP130 AP147 AP164 AP181 AP198 AP215 AP232 AP249 A12 AP12 AP29AP46 AP63 AP80 AP97 AP114 AP131 AP148 AP165 AP182 AP199 AP216 AP233AP250 A13 AP13 AP30 AP47 AP64 AP81 AP98 AP115 AP132 AP149 AP166 AP183AP200 AP217 AP234 AP251 A14 AP14 AP31 AP48 AP65 AP82 AP99 AP116 AP133AP150 AP167 AP184 AP201 AP218 AP235 AP252 A15 AP15 AP32 AP49 AP66 AP83AP100 AP117 AP134 AP151 AP168 AP185 AP202 AP219 AP236 AP253 A16 AP16AP33 AP50 AP67 AP84 AP101 AP118 AP135 AP152 AP169 AP186 AP203 AP220AP237 AP254 A17 AP17 AP34 AP51 AP68 AP85 AP102 AP119 AP136 AP153 AP170AP187 AP204 AP221 AP238 AP255 †ApoA-1 Variant Combinations A1-A17 areshown in Table 3. ‡PON1 Variant Combinations P1-P15 are shown in Table4.

In yet other embodiments comprising a second polypeptide segmentcarboxyl-terminal to the first polypeptide segment (e.g.,carboxyl-terminal to a dimerizing domain), the second polypeptidesegment is a platelet-activating factor acetylhydrolase (PAF-AH). Insome embodiments, the platelet-activating factor acetylhydrolase is ahuman PAF-AH or a functional variant or fragment thereof. For example,in some embodiments, the second polypeptide segment comprises an aminoacid sequence having at least 80% identity with amino acid residues22-441 of SEQ ID NO:32. In more particular embodiments, the secondpolypeptide segment comprises an amino acid sequence having at least85%, at least 90%, or at least 95% identity with amino acid residues22-441 of SEQ ID NO:32. In yet other embodiments, the second polypeptidesegment comprises an amino acid sequence having at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity with aminoacid residues 22-441 of SEQ ID NO:32.

In still other embodiments comprising a second polypeptide segmentcarboxyl-terminal to the first polypeptide segment (e.g.,carboxyl-terminal to a dimerizing domain), the second polypeptidesegment is a cholesterol ester transfer protein (CETP). In someembodiments, the cholesterol ester transfer protein is a human CETP or afunctional variant or fragment thereof. For example, in someembodiments, the second polypeptide segment comprises an amino acidsequence having at least 80% identity with amino acid residues 18-493 ofSEQ ID NO:30. In more particular embodiments, the second polypeptidesegment comprises an amino acid sequence having at least 85%, at least90%, or at least 95% identity with amino acid residues 18-493 of SEQ IDNO:30. In yet other embodiments, the second polypeptide segmentcomprises an amino acid sequence having at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity with amino acidresidues 18-493 of SEQ ID NO:30.

Polypeptide linkers for use in accordance with the present invention canbe naturally-occurring, synthetic, or a combination of both. The linkerjoins two separate polypeptide regions (e.g., a dimerizing domain and anApoA-1 polypeptide) and maintains the linked polypeptide regions asseparate and discrete domains of a longer polypeptide. The linker canallow the separate, discrete domains to cooperate yet maintain separateproperties (e.g., in the case of an Fc region dimerizing domain linkedto an ApoA-1 polypeptide, Fc receptor (e.g., FcRn) binding may bemaintained for the Fc region, while functional properties of the ApoA-1polypeptide (e.g., lipid binding) will be maintained). For examples ofthe use of naturally occurring as well as artificial peptide linkers toconnect heterologous polypeptides, see, e.g., Hallewell et al., J. Biol.Chem. 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731,1995; Robinson and Sauer, Biochemistry 35, 109-116, 1996; Khandekar etal., J. Biol. Chem. 272, 32190-32197, 1997; Fares et al., Endocrinology139, 2459-2464, 1998; Smallshaw et al., Protein Eng. 12, 623-630, 1999;U.S. Pat. No. 5,856,456.

Typically, residues within the linker polypeptide are selected toprovide an overall hydrophilic character and to be non-immunogenic andflexible. As used herein, a “flexible” linker is one that lacks asubstantially stable higher-order conformation in solution, althoughregions of local stability are permissible. In general, small, polar,and hydrophilic residues are preferred, and bulky and hydrophobicresidues are undesirable. Areas of local charge are to be avoided; ifthe linker polypeptide includes charged residues, they will ordinarilybe positioned so as to provide a net neutral charge within a smallregion of the polypeptide. It is therefore preferred to place a chargedresidue adjacent to a residue of opposite charge. In general, preferredresidues for inclusion within the linker polypeptide include Gly, Ser,Ala, Thr, Asn, and Gln; more preferred residues include Gly, Ser, Ala,and Thr; and the most preferred residues are Gly and Ser. In general,Phe, Tyr, Trp, Pro, Leu, Ile, Lys, and Arg residues will be avoided(unless present within an immunoglobulin hinge region of the linker),Pro residues due to their hydrophobicity and lack of flexibility, andLys and Arg residues due to potential immunogenicity. The sequence ofthe linker will also be designed to avoid unwanted proteolysis.

In certain embodiments, linker L1 comprises at least two or at leastthree amino acid residues. In some embodiments, L1 comprises at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 20, at least 26, or at least 36amino acid residues. In particular variations, L1 consists of from twoto 60, from two to 50, from two to 40, from two to 36, from two to 35,from two to 31, from two to 30, from two to 26, from three to 60, fromthree to 50, from three to 40, from three to 36, from three to 35, fromthree to 31, from three to 30, from three to 26, from four to 60, fromfour to 50, from four to 40, from four to 36, from four to 35, from fourto 31, from four to 30, or from four to 26 amino acid residues. In othervariations, L1 consists of from five to 60, from five to 50, from fiveto 40, from five to 36, from five to 35, from five to 31, from five to30, from five to 26, from six to 60, from six to 50, from six to 40,from six to 36, from six to 35, from six to 31, from six to 30, from sixto 26, from seven to 60, from seven to 50, from seven to 40, from sevento 36, from seven to 35, from seven to 31, from seven to 30, or fromseven to 26 amino acid residues. In other variations, L1 consists offrom eight to 60, from eight to 50, from eight to 40, from eight to 36,from eight to 35, from eight to 31, from eight to 30, from eight to 26,from nine to 60, from nine to 50, from nine to 40, from nine to 36, fromnine to 35, from nine to 31, from nine to 30, from nine to 26, from 10to 60, from 10 to 50, from 10 to 40, from 10 to 36, from 10 to 35, from10 to 31, from 10 to 30, or from 10 to 26 amino acid residues. In othervariations, L1 consists of from 11 to 60, from 11 to 50, from 11 to 40,from 11 to 36, from 11 to 35, from 11 to 31, from 11 to 30, from 11 to26, from 12 to 60, from 12 to 50, from 12 to 40, from 12 to 36, from 12to 35, from 12 to 31, from 12 to 30, from 12 to 26, from 15 to 60, from15 to 40, from 15 to 50, from 15 to 36, from 15 to 35, from 15 to 31,from 15 to 30, or from 15 to 26 amino acid residues. In othervariations, L1 consists of from 16 to 60, from 16 to 50, from 16 to 40,or from 16 to 36 amino acid residues. In yet other variations, L1consists of from 20 to 60, from 20 to 50, from 20 to 40, from 20 to 36,from 25 to 60, from 25 to 50, from 25 to 40, or from 25 to 36 amino acidresidues. In still other variations, L1 consists of from 26 to 60, from26 to 50, from 26 to 40, or from 26 to 36 amino acid residues. In morespecific variations, L1 consists of 16 amino acid residues, 21 aminoacid residues, 26 amino acid residues, 31 amino acid residues, or 36amino acid residues. In some embodiments, L1 comprises or consists ofthe amino acid sequence shown in residues 268-293 of SEQ ID NO:2,residues 268-288 of SEQ ID NO:26, residues 268-283 of SEQ ID NO:22, SEQID NO:54, or residues 268-303 of SEQ ID NO:24.

Exemplary L2 linkers comprise at least three amino acid residues and aretypically up to 60 amino acid residues. In certain variations, L2linkers have a range of sequence lengths as described above for L1. In aspecific embodiment of a polypeptide comprising the formulaApoA1-L1-D-L2-P and where L2 is present and P is an RNase, L2 comprisesor consists of the amino acid sequence shown in residues 526-543 of SEQID NO:4.

In certain embodiments, polypeptide linkers comprise a plurality ofglycine residues. For example, in some embodiments, a polypeptide linker(e.g., L1) comprises a plurality of glycine residues and optionally atleast one serine residue. In particular variations, a polypeptide linker(e.g., L1) comprises the sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:15),such as, e.g., two or more tandem repeats of the amino acid sequence ofSEQ ID NO:15. In some embodiments, a linker comprises the sequence[Gly-Gly-Gly-Gly-Ser]_(n) ([SEQ ID NO:15]_(n)), where n is a positiveinteger such as, for example, an integer from 1 to 5, from 2 to 5, from3 to 5, from 1 to 6, from 2 to 6, from 3 to 6, or from 4 to 6. In aspecific variation of a polypeptide linker comprising the formula[Gly-Gly-Gly-Gly-Ser]_(n), n is 4. In another specific variation of apolypeptide linker comprising the formula [Gly-Gly-Gly-Gly-Ser]_(n), nis 3. In yet another specific variation of a polypeptide linkercomprising the formula [Gly-Gly-Gly-Gly-Ser]_(n), n is 5. In stillanother specific variation of a polypeptide linker comprising theformula [Gly-Gly-Gly-Gly-Ser]_(n), n is 6. In certain embodiments, apolypeptide linker comprises a series of glycine and serine residues(e.g., [Gly-Gly-Gly-Gly-Ser]_(n), where n is defined as above) insertedbetween two other sequences of the polypeptide linker (e.g., any of thepolypeptide linker sequences described herein). In other embodiments, apolypeptide linker includes glycine and serine residues (e.g.,[Gly-Gly-Gly-Gly-Ser]_(n), where n is defined as above) attached at oneor both ends of another sequence of the polypeptide linker (e.g., any ofthe polypeptide linker sequences described herein). In one embodiment, apolypeptide linker comprises at least a portion of an upper hinge region(e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule), at least aportion of a middle hinge region (e.g., derived from an IgG1, IgG2,IgG3, or IgG4 molecule) and a series of glycine and serine amino acidresidues (e.g., [Gly-Gly-Gly-Gly-Ser]_(n), wherein n is defined asabove).

In another embodiment, a polypeptide linker comprises a non-naturallyoccurring immunoglobulin hinge region, e.g., a hinge region that is notnaturally found in an immunoglobulin and/or a hinge region that has beenaltered so that it differs in amino acid sequence from a naturallyoccurring immunoglobulin hinge region. In one embodiment, mutations canbe made to a hinge region to make a polypeptide linker. In oneembodiment, a polypeptide linker comprises a hinge domain that does notcomprise a naturally occurring number of cysteines, i.e., thepolypeptide linker comprises either fewer cysteines or a greater numberof cysteines than a naturally occurring hinge molecule.

Various dimerization domains are suitable for use in accordance with thefusion polypeptides and dimeric fusion proteins as described herein. Incertain embodiments, the dimerizing domain is an immunoglobulin heavychain constant region, such as an Fc region. The Fc region may be anative sequence Fc region or a variant Fc region. In some embodiments,the Fc region lacks one or more effector functions (e.g., one or both ofADCC and CDC effector functions).

In some embodiments, the dimerizing domain is an Fc region of a humanantibody with a mutation in the CH2 region so that the molecule is notglycosylated, including but not limited to N297 (EU numbering for humanIgG heavy chain constant region) (corresponding to amino acid position375 of SEQ ID NO:2). In another embodiment, the Fc region is human IgG1(yl) with the three cysteines of the hinge region (C220, C226, C229)each changed to serine, and the proline at position 238 of the CH2domain changed to serine. In another preferred embodiment, the Fc regionis human γ1 with N297 changed to any other amino acid. In anotherembodiment, the Fc region is human γ1 with one or more amino acidsubstitutions between Eu positions 292 and 300. In another embodiment,the Fc region is human γ1 with one or more amino acid additions ordeletions at any position between residues 292 and 300. In anotherembodiment, the Fc region is human γ1 with an SCC hinge (i.e., withcysteine C220 changed to serine and with a cysteine at each of Eupositions 226 and 229) or an SSS hinge (i.e., each of the threecysteines at Eu positions 220, 226, and 229 changed to serine). Infurther embodiments, the Fc region is human γ1 with an SCC hinge and aP238 mutation. In another embodiment, the Fc domain is human γ1 withmutations that alter binding by Fc gamma receptors (I, II, III) withoutaffecting FcRn binding important for half-life. In further embodiments,an Fc region is as disclosed in Ehrhardt and Cooper, Curr. Top.Microbiol. Immunol. 2010 Aug. 3 (Immunoregulatory Roles for FcReceptor-Like Molecules); Davis et al., Ann. Rev. Immunol. 25:525-60,2007 (Fc receptor-like molecules); or Swainson et al., J. Immunol.184:3639-47, 2010.

In certain embodiments, an Fc region is a human IgG variant (e.g., ahuman γ1 variant) in which one or more of the cysteine residues in thehinge region have each been changed to a non-cysteine residue. Forexample, in some embodiments, the Fc region is a human IgG variant inwhich all of the cysteine residues in the hinge region have each beenchanged to a non-cysteine residue. A particularly suitable Fc region isa human γ1 variant in which each of the three cysteines at Eu positions220, 226, and 229 changed; in some such variations, each cysteine isreplaced with serine.

In some embodiments of a fusion polypeptide comprising an Fc dimerizingdomain, the Fc region comprises an amino acid substitution that altersthe antigen-independent effector functions of the fusion protein. Insome such embodiments, the Fc region includes an amino acid substitutionthat alters the circulating half-life of the resulting molecule. Suchantibody derivatives exhibit either increased or decreased binding toFcRn when compared to antibodies lacking these substitutions and,therefore, have an increased or decreased half-life in serum,respectively. Fc variants with improved affinity for FcRn areanticipated to have longer serum half-lives, and such antibodies haveuseful applications in methods of treating mammals where long half-lifeof the administered antibody is desired. In contrast, Fc variants withdecreased FcRn binding affinity are expected to have shorter half-lives,and such antibodies are also useful, for example, for administration toa mammal where a shortened circulation time may be advantageous, e.g.,where the starting antibody has toxic side effects when present in thecirculation for prolonged periods. Fc variants with decreased FcRnbinding affinity are also less likely to cross the placenta and, thus,are also useful in the treatment of diseases or disorders in pregnantwomen. In addition, other applications in which reduced FcRn bindingaffinity may be desired include those applications in which localizationto the brain, kidney, and/or liver is desired. In one exemplaryembodiment, the antibodies of the invention exhibit reduced transportacross the epithelium of kidney glomeruli from the vasculature. Inanother embodiment, the fusion proteins of the invention exhibit reducedtransport across the blood brain barrier (BBB) from the brain, into thevascular space. In one embodiment, a fusion protein with altered FcRnbinding comprises an Fc region having one or more amino acidsubstitutions within the “FcRn binding loop” of the Fc domain. Exemplaryamino acid substitutions which altered FcRn binding activity aredisclosed in International PCT Publication No. WO 05/047327, which isincorporated by reference herein.

In other embodiments, a fusion polypeptide of the present inventioncomprises an Fc variant comprising an amino acid substitution whichalters the antigen-dependent effector functions of the polypeptide, inparticular ADCC or complement activation, e.g., as compared to a wildtype Fc region. In an exemplary embodiment, such fusion polypeptidesexhibit altered binding to an Fc gamma receptor (FcγR, e.g., CD16). Suchfusion polypeptides exhibit either increased or decreased binding toFcγR when compared to wild-type polypeptides and, therefore, mediateenhanced or reduced effector function, respectively. Fc variants withimproved affinity for FcγRs are anticipated to enhance effectorfunction, and such fusion proteins have useful applications in methodsof treating mammals where target molecule destruction is desired. Incontrast, Fc variants with decreased FcγR binding affinity are expectedto reduce effector function, and such fusion proteins are also useful,for example, for treatment of conditions in which target celldestruction is undesirable, e.g., where normal cells may express targetmolecules, or where chronic administration of the antibody might resultin unwanted immune system activation. In one embodiment, the fusionpolypeptide comprising an Fc region exhibits at least one alteredantigen-dependent effector function selected from the group consistingof opsonization, phagocytosis, complement dependent cytotoxicity,antigen-dependent cellular cytotoxicity (ADCC), or effector cellmodulation as compared to a polypeptide comprising a wild-type Fcregion.

In one embodiment, a fusion polypeptide comprising an Fc region exhibitsaltered binding to an activating FcγR (e.g., FcγI, FcγHa, or FcγRIIIa).In another embodiment, the fusion protein exhibits altered bindingaffinity to an inhibitory FcγR (e.g., FcγRIIb). Exemplary amino acidsubstitutions which altered FcR or complement binding activity aredisclosed in International PCT Publication No. WO 05/063815, which isincorporated by reference herein.

A fusion polypeptide comprising an Fc region may also comprise an aminoacid substitution that alters the glycosylation of the Fc region. Forexample, the Fc domain of the fusion protein may have a mutation leadingto reduced glycosylation (e.g., N- or O-linked glycosylation) or maycomprise an altered glycoform of the wild-type Fc domain (e.g., a lowfucose or fucose-free glycan). In another embodiment, the molecule hasan amino acid substitution near or within a glycosylation motif, forexample, an N-linked glycosylation motif that contains the amino acidsequence NXT or NXS. Exemplary amino acid substitutions which reduce oralter glycosylation are disclosed in International PCT Publication No.WO 05/018572 and US Patent Application Publication No. 2007/0111281,which are incorporated by reference herein.

It will be understood by those of skill in the art that variousembodiments of Fc variants as described herein can be combined in thefusion polypeptides of the present invention, unless the context clearlyindicates otherwise.

In some embodiments, a dimerizing domain is an Fc region comprising anamino acid sequence having at least 80%, at least 85%, at least 90%, orat least 95% identity with an amino acid sequence selected from sequenceshown in (i) residues 294-525 or 294-524 of SEQ ID NO:2, or (ii)residues 294-525 or 294-524 of SEQ ID NO:13. In yet other embodiments,the Fc region comprises an amino acid sequence having at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe amino acid sequence shown in (i) residues 294-525 or 294-524 of SEQID NO:2, or (ii) residues 294-525 or 294-524 of SEQ ID NO:13.

In some embodiments of a fusion polypeptide comprising ApoA1-L1-D asdescribed above, the fusion polypeptide comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, or at least 95%identity with an amino acid sequence selected from sequence shown in (i)residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii) residues19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13, (iii) residues19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20, (iv) residues 19-515,19-514, 25-515, or 25-514 of SEQ ID NO:22, (v) residues 19-520, 19-519,25-520, or 25-519 of SEQ ID NO:26, or (vi) residues 19-535, 19-534,25-535, or 25-534 of SEQ ID NO:24. In yet other embodiments, the fusionpolypeptide comprises an amino acid sequence having at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity withthe amino acid sequence shown in (i) residues 19-525, 19-524, 25-525, or25-524 of SEQ ID NO:2, (ii) residues 19-525, 19-524, 25-525, or 25-524of SEQ ID NO:13, (iii) residues 19-501, 19-500, 25-501, or 25-500 of SEQID NO:20, (iv) residues 19-515, 19-514, 25-515, or 25-514 of SEQ IDNO:22, (v) residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26,or (vi) residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24. Insome embodiments, the fusion polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, or at least95% identity with (i) residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:2, (ii) residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:13, (iii) residues 19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20,(iv) residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22, (v)residues 19-520, 19-519, 25-520, or 25-519 of SEQ ID NO:26, or (vi)residues 19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24, where thefusion polypeptide comprises at least one amino acid substitution in thefirst (“ApoA1”) polypeptide segment selected from V156[E/K],Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F,and W132F as described herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] andoptionally at least one of M86[L/I/V], M112[L/I/V], and M148[L/I/V];both V156[E/K] and Y192[S/Q/N/H/F] and optionally at least one ofM86[L/I/V], M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F,W72F, and W132F); in some such embodiments, the fusion polypeptidecomprises an amino acid sequence that is otherwise 100% identical to (i)residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii) residues19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13, (iii) residues19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20, (iv) residues 19-515,19-514, 25-515, or 25-514 of SEQ ID NO:22, (v) residues 19-520, 19-519,25-520, or 25-519 of SEQ ID NO:26, or (vi) residues 19-535, 19-534,25-535, or 25-534 of SEQ ID NO:24. In some variations, the fusionpolypeptide comprises an amino acid sequence having at least 80%, atleast 85%, at least 90%, or at least 95% identity with an amino acidsequence selected from sequence shown in (i) residues 19-525, 19-524,25-525, or 25-524 of SEQ ID NO:2, (ii) residues 19-525, 19-524, 25-525,or 25-524 of SEQ ID NO:13, (iii) residues 19-501, 19-500, 25-501, or25-500 of SEQ ID NO:20, (iv) residues 19-515, 19-514, 25-515, or 25-514of SEQ ID NO:22, (v) residues 19-520, 19-519, 25-520, or 25-519 of SEQID NO:26, or (vi) residues 19-535, 19-534, 25-535, or 25-534 of SEQ IDNO:24, where the fusion polypeptide comprises, in the ApoA1 polypeptidesegment, the specific V156[E/K] and/or Y192[S/Q/N/H/F] substitution(s)of any one of variant combinations A1-A17 as shown in Table 3 herein; insome such embodiments, the fusion polypeptide comprises an amino acidsequence that is otherwise 100% identical to (i) residues 19-525,19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii) residues 19-525, 19-524,25-525, or 25-524 of SEQ ID NO:13, (iii) residues 19-501, 19-500,25-501, or 25-500 of SEQ ID NO:20, (iv) residues 19-515, 19-514, 25-515,or 25-514 of SEQ ID NO:22, (v) residues 19-520, 19-519, 25-520, or25-519 of SEQ ID NO:26, or (vi) residues 19-535, 19-534, 25-535, or25-534 of SEQ ID NO:24.

In some embodiments of a fusion polypeptide comprising ApoA1-L1-D-L2-Pas described above and where P is an RNase, the fusion polypeptidecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, or at least 95% identity with the amino acid sequence shownin (i) residues 19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or25-675 of SEQ ID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58,or (iv) residues 19-671 or 25-671 of SEQ ID NO:59. In yet otherembodiments, the fusion polypeptide comprises an amino acid sequencehaving at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the amino acid sequence shown in (i) residues19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or 25-675 of SEQID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or (iv)residues 19-671 or 25-671 of SEQ ID NO:59. In some embodiments, thefusion polypeptide comprises an amino acid sequence having at least 80%,at least 85%, at least 90%, or at least 95% identity with (i) residues19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or 25-675 of SEQID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or (iv)residues 19-671 or 25-671 of SEQ ID NO:59, where the fusion polypeptidecomprises at least one amino acid substitution in the first (“ApoA1”)polypeptide segment selected from V156[E/K], Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F, and W132F asdescribed herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; both V156[E/K]and Y192[S/Q/N/H/F] and optionally at least one of M86[L/I/V],M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F, W72F, andW132F); in some such embodiments, the fusion polypeptide comprises anamino acid sequence that is otherwise 100% identical to (i) residues19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or 25-675 of SEQID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or (iv)residues 19-671 or 25-671 of SEQ ID NO:59. In some variations, thefusion polypeptide comprises an amino acid sequence having at least 80%,at least 85%, at least 90%, or at least 95% identity with an amino acidsequence selected from sequence shown in (i) residues 19-675 or 25-675of SEQ ID NO:4, (ii) residues 19-675 or 25-675 of SEQ ID NO:14, (iii)residues 19-671 or 25-671 of SEQ ID NO:58, or (iv) residues 19-671 or25-671 of SEQ ID NO:59, where the fusion polypeptide comprises, in theApoA1 polypeptide segment, the specific V156[E/K] and/or Y192[S/Q/N/H/F]substitution(s) of any one of variant combinations A1-A17 as shown inTable 3 herein; in some such embodiments, the fusion polypeptidecomprises an amino acid sequence that is otherwise 100% identical to (i)residues 19-675 or 25-675 of SEQ ID NO:4, (ii) residues 19-675 or 25-675of SEQ ID NO:14, (iii) residues 19-671 or 25-671 of SEQ ID NO:58, or(iv) residues 19-671 or 25-671 of SEQ ID NO:59.

In some embodiments of a fusion polypeptide comprising ApoA1-L1-D-L2-Pas described above and where P is a paraoxonase, the fusion polypeptidecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, or at least 95% identity with the amino acid sequence shownin (i) residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873or 25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ IDNO:46, or (iv) residues 19-883 or 25-883 of SEQ ID NO:48. In yet otherembodiments, the fusion polypeptide comprises an amino acid sequencehaving at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity with the amino acid sequence shown in (i) residues19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46, or (iv)residues 19-883 or 25-883 of SEQ ID NO:48. In some embodiments, thefusion polypeptide comprises an amino acid sequence having at least 80%,at least 85%, at least 90%, or at least 95% identity with (i) residues19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46, or (iv)residues 19-883 or 25-883 of SEQ ID NO:48, where the fusion polypeptidecomprises (A) at least one amino acid substitution in the first(“ApoA1”) polypeptide segment selected from V156[E/K], Y192[S/Q/N/H/F],M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F, and W132F asdescribed herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; both V156[E/K]and Y192[S/Q/N/H/F] and optionally at least one of M86[L/I/V],M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F, W72F, andW132F), and/or (B) at least one substitution in the second (“P”)polypeptide segment selected from Y185[H/Q/S] and F293[H/Q/N] asdescribed herein; in some such embodiments, the fusion polypeptidecomprises an amino acid sequence that is otherwise 100% identical to (i)residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46,or (iv) residues 19-883 or 25-883 of SEQ ID NO:48; in particularvariations, the fusion polypeptide comprises substitutions of both (A)and (B) as above (e.g., V156[E/K] and/or Y192[S/Q/N/H/F] and optionallyat least one of M86[L/I/V], M112[L/I/V], and M148[L/I/V] in the firstpolypeptide segment and Y185[H/Q/S] and optionally F293[H/Q/N] in thesecond polypeptide segment). In some embodiments, the fusion polypeptidecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, or at least 95% identity with (i) residues 19-883 or 25-883of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQ ID NO:38, (iii)residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) residues 19-883 or25-883 of SEQ ID NO:48, where the fusion polypeptide comprises (A) inthe ApoA1 polypeptide segment, the specific V156[E/K] and/orY192[S/Q/N/H/F] substitution(s) of any one of variant combinationsA1-A17 as shown in Table 3 herein, and/or (B) in the P polypeptidesegment, the specific Y185[H/Q/S] and/or F293[H/Q/N] substitution(s) ofany one of variant combinations P1-P15 as shown in Table 4 herein (e.g.,a fusion polypeptide comprising a specific combination of variantsselected from combinations AP1-AP255 as shown in Table 5 herein); insome such embodiments, the fusion polypeptide comprises an amino acidsequence that is otherwise 100% identical to (i) residues 19-883 or25-883 of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQ ID NO:38,(iii) residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) residues 19-883or 25-883 of SEQ ID NO:48.

In some embodiments of a fusion polypeptide comprising ApoA1-L1-D-L2-Pas described above and where P is a platelet-activating factoracetylhydrolase (PAF-AH), the fusion polypeptide comprises an amino acidsequence having at least 80%, at least 85%, at least 90%, or at least95% identity with the amino acid sequence shown in residues 19-963 or25-963 of SEQ ID NO:34. In yet other embodiments, the fusion polypeptidecomprises an amino acid sequence having at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity with the amino acidsequence shown in residues 19-963 or 25-963 of SEQ ID NO:34. In someembodiments, the fusion polypeptide comprises an amino acid sequencehaving at least 80%, at least 85%, at least 90%, or at least 95%identity with residues 19-963 or 25-963 of SEQ ID NO:34, where thefusion polypeptide comprises at least one amino acid substitution in thefirst (“ApoA1”) polypeptide segment selected from V156[E/K],Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F,and W132F as described herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] andoptionally at least one of M86[L/I/V], M112[L/I/V], and M148[L/I/V];both V156[E/K] and Y192[S/Q/N/H/F] and optionally at least one ofM86[L/I/V], M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F,W72F, and W132F); in some such embodiments, the fusion polypeptidecomprises an amino acid sequence that is otherwise 100% identical toresidues 19-963 or 25-963 of SEQ ID NO:34. In some variations, thefusion polypeptide comprises an amino acid sequence having at least 80%,at least 85%, at least 90%, or at least 95% identity with an amino acidsequence selected from sequence shown in residues 19-963 or 25-963 ofSEQ ID NO:34, where the fusion polypeptide comprises, in the ApoA1polypeptide segment, the specific V156[E/K] and/or Y192[S/Q/N/H/F]substitution(s) of any one of variant combinations A1-A17 as shown inTable 3 herein; in some such embodiments, the fusion polypeptidecomprises an amino acid sequence that is otherwise 100% identical toresidues 19-963 or 25-963 of SEQ ID NO:34.

In some embodiments of a fusion polypeptide comprising ApoA1-L1-D-L2-Pas described above and where P is a cholesterol ester transfer protein(CETP), the fusion polypeptide comprises an amino acid sequence havingat least 80%, at least 85%, at least 90%, or at least 95% identity withthe amino acid sequence shown in residues 19-1019 or 25-1019 of SEQ IDNO:40. In yet other embodiments, the fusion polypeptide comprises anamino acid sequence having at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity with the amino acid sequence shownin residues 19-1019 or 25-1019 of SEQ ID NO:40. In some embodiments, thefusion polypeptide comprises an amino acid sequence having at least 80%,at least 85%, at least 90%, or at least 95% identity with residues19-1019 or 25-1019 of SEQ ID NO:40, where the fusion polypeptidecomprises at least one amino acid substitution in the first (“ApoA1”)polypeptide segment selected from V156[E/K], Y192[S/Q/N/H/F],M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F, and W132F asdescribed herein (e.g., V156[E/K] or Y192[S/Q/N/H/F] and optionally atleast one of M86[L/I/V], M112[L/I/V], and M148[L/I/V]; both V156[E/K]and Y192[S/Q/N/H/F] and optionally at least one of M86[L/I/V],M112[L/I/V], and M148[L/I/V]; or all four of W8F, W50F, W72F, andW132F); in some such embodiments, the fusion polypeptide comprises anamino acid sequence that is otherwise 100% identical to residues 19-1019or 25-1019 of SEQ ID NO:40. In some variations, the fusion polypeptidecomprises an amino acid sequence having at least 80%, at least 85%, atleast 90%, or at least 95% identity with an amino acid sequence selectedfrom sequence shown in residues 19-1019 or 25-1019 of SEQ ID NO:40,where the fusion polypeptide comprises, in the ApoA1 polypeptidesegment, the specific V156[E/K] and/or Y192[S/Q/N/H/F] substitution(s)of any one of variant combinations A1-A17 as shown in Table 3 herein; insome such embodiments, the fusion polypeptide comprises an amino acidsequence that is otherwise 100% identical to residues 19-1019 or 25-1019of SEQ ID NO:40.

The present invention also provides dimeric proteins comprising firstand second polypeptide fusions as described above. Accordingly, inanother aspect, the present invention provides a dimeric proteincomprising a first fusion polypeptide and a second fusion polypeptide,where each of the first and second polypeptide fusions comprises, froman amino-terminal position to a carboxyl-terminal position, ApoA1-L1-D,where ApoA1 is a first polypeptide segment having cholesterol effluxactivity and which is selected from (i) a naturally occurring ApoA-1polypeptide or a functional variant or fragment thereof and (ii) anApoA-1 mimetic; L1 is a first polypeptide linker; and D is a dimerizingdomain. In some embodiments, each of the first and second fusionpolypeptides further includes a second polypeptide segment locatedcarboxyl-terminal to the dimerizing domain. In particular variations,the second polypeptide segment is (a) a naturally occurring RNase,paraoxonase, platelet-activating factor acetylhydrolase (PAF-AH),cholesterol ester transfer protein (CETP), or lecithin-cholesterolacyltransferase (LCAT); (b) a functional variant or fragment of any ofthe naturally occurring proteins specified in (a); (c) a polypeptidethat specifically binds to proprotein convertase subtilisin/kexin type 9(PCSK9) and inhibits PCSK9 activity such as, e.g., a PCSK9-specificscFv; or (d) a polypeptide that specifically binds to amyloid beta (Aβ)such as, e.g., an Aβ-specific scFv. Such a fusion polypeptide comprisinga second polypeptide segment may be represented by the formulaApoA1-L1-D-L2-P (from an amino-terminal position to a carboxyl-terminalposition), where ApoA1, L1, and D are each as previously defined, whereL2 is a second polypeptide linker and is optionally present, and where Pis the second polypeptide segment.

In another aspect, the present invention provides a dimeric proteincomprising a first fusion polypeptide and a second fusion polypeptide,where each of the first and second fusion polypeptides comprises a firstpolypeptide segment, a second polypeptide segment, and a dimerizingdomain, where the first polypeptide segment has cholesterol effluxactivity and is selected from (i) a naturally occurring ApoA-1polypeptide or a functional variant or fragment thereof and (ii) anApoA-1 mimetic, and where the second polypeptide segment is locatedcarboxyl-terminal to the first polypeptide segment and is (a) anaturally occurring RNase, paraoxonase, platelet-activating factoracetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), orlecithin-cholesterol acyltransferase (LCAT), (b) a functional variant orfragment of any of the naturally occurring proteins specified in (a),(c) a polypeptide that specifically binds to proprotein convertasesubtilisin/kexin type 9 (PCSK9) and inhibits PCSK9 activity such as,e.g., a PCSK9-specific scFv, or (d) a polypeptide that specificallybinds to amyloid beta (Aβ) such as, e.g., an Aβ-specific scFv. In someembodiments, the dimerizing domain is located carboxyl-terminal to thefirst polypeptide segment and amino-terminal to the second polypeptidesegment.

In another aspect, the present invention provides (a) a first fusionpolypeptide comprising an immunoglobulin heavy chain linkedcarboxyl-terminal to an ApoA-1 polypeptide or ApoA-1 mimetic and (b) asecond fusion polypeptide comprising an immunoglobulin light chainlinked carboxyl-terminal to the ApoA-1 polypeptide or ApoA-1 mimetic.The first and second fusion polypeptides can be co-expressed to create astable tetramer composed of two double belt ApoA-1 dimers, whereinlinkers between ApoA-1 and the heavy chain and between ApoA-1 and thelight chain are of sufficient length to allow cholesterol efflux andreverse cholesterol transport.

The fusion polypeptides of the present invention, including dimericfusion proteins, can further be conjugated to an effector moiety. Theeffector moiety can be any number of molecules, including, e.g., alabeling moiety such as a radioactive label or fluorescent label, a TLRligand or binding domain, an enzyme, or a therapeutic moiety. In aparticular embodiment, the effector moiety is a myeloperoxidase (MPO)inhibitor. MPO inhibitors are generally known (see, e.g., Malle et al.,Br J Pharmacol. 152: 838-854, 2007) and may be readily conjugated tofusion polypeptides as described herein. Exemplary MPO inhibitorsinclude inhibitors based on 3-alkylindole derivatives (see Soubhye etal., J Med Chem 56:3943-58, 2013; describing studies of 3-alkylindolederivatives as selective and highly potent myeloperoxidase inhibitors,including a compound with high and selective inhibition of MPO (IC50=18nM)); inhibitors based on 3-(aminoalkyl)-5-fluorindoles (see Soubhye etal., J Med Chem 53: 8747-8759, 2010); inhibitors based on 2H-indazolesand 1H-indazolones (see Roth et al., Bioorg Med Chem 22: 6422-6429,2014; describing the evaluation 2H-indazoles and 1H-indazolones and theidentification of compounds with IC50 values <1 μM); and benzoic acidhydrazide-containing compounds (see Huang et al., Arch Biochem Biophys570: 14-22, 2015; showing inactivation of MPO by benzoic acidhydrazide-containing compounds, where the light chain subunit of MPO isfreed from the larger heavy chain by cleavage of the ester bond).

In another embodiment, the fusion polypeptides of the present invention,including dimeric fusion proteins, are modified to extend half-life,such as, for example, by attaching at least one molecule to the fusionprotein for extending serum half-life. Such molecules for attachment mayinclude, e.g., a polyethlyene glycol (PEG) group, serum albumin,transferrin, transferrin receptor or the transferrin-binding portionthereof, or a combination thereof. Methods for such modification aregenerally well-known in the art. As used herein, the word “attached”refers to a covalently or noncovalently conjugated substance. Theconjugation may be by genetic engineering or by chemical means.

III. Materials and Methods for Making Polypeptide Fusions and DimericProteins

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the fusion polypeptides disclosedabove. The polynucleotides of the present invention include bothsingle-stranded and double-stranded molecules. Polynucleotides encodingvarious segments of a fusion polypeptide (e.g., a dimerizing domain suchas an Fc fragment; ApoA1 and P polypeptide segments) can be generatedand linked together to form a polynucleotide encoding a fusionpolypeptide as described herein using known methods for recombinantmanipulation of nucleic acids.

DNA sequences encoding ApoA-1, RNases (e.g., RNase 1), paraoxonases(e.g., PON1), platelet-activating factor acetylhydrolase (PAF-AH),cholesterol ester transfer protein (CETP), and lecithin-cholesterolacyltransferase (LCAT) are known in the art. DNA sequences encodingvarious dimerizing domains (e.g., immunoglobulin heavy chain constantregions such as Fc fragments) are also known. Polynucleotides encoding,e.g., the variable regions of PCSK9-binding or Aβ-binding antibodies,including scFvs, are also readily identifiable using techniqueswell-known in the art such as screening of recombinant antibodyexpression libraries (e.g., phage display expression libraries).Additional DNA sequences encoding any of these polypeptides can bereadily generated by those of ordinary skill in the art based on thegenetic code. Counterpart RNA sequences can be generated by substitutionof U for T. Those skilled in the art will readily recognize that, inview of the degeneracy of the genetic code, considerable sequencevariation is possible among polynucleotide molecules encoding a givenpolypeptide. DNA and RNA encoding functional variants and fragments ofsuch polypeptides can also be obtained using known recombinant methodsto introduce variation into a polynucleotide sequence, followed byexpression of the encoded polypeptide and determination of functionalactivity (e.g., cholesterol efflux) using an appropriate screeningassay.

Methods for preparing DNA and RNA are well known in the art. Forexample, complementary DNA (cDNA) clones can be prepared from RNA thatis isolated from a tissue or cell that produces large amounts of RNAencoding a polypeptide of interest. Total RNA can be prepared usingguanidine HCl extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly(A)±RNA is prepared from total RNA using the method of Aviv and Leder(Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA isprepared from poly(A)⁺ RNA using known methods. In the alternative,genomic DNA can be isolated. Methods for identifying and isolating cDNAand genomic clones are well known and within the level of ordinary skillin the art, and include the use of the sequences disclosed herein, orparts thereof, for probing or priming a library. Polynucleotidesencoding polypeptides of interest are identified and isolated by, forexample, hybridization or polymerase chain reaction (“PCR,” Mullis, U.S.Pat. No. 4,683,202). Expression libraries can be probed with antibodiesto the polypeptide of interest, receptor fragments, or other specificbinding partners.

The polynucleotides of the present invention can also be prepared byautomated synthesis. The production of short, double-stranded segments(60 to 80 bp) is technically straightforward and can be accomplished bysynthesizing the complementary strands and then annealing them. Longersegments (typically >300 bp) are assembled in modular form fromsingle-stranded fragments that are from 20 to 100 nucleotides in length.Automated synthesis of polynucleotides is within the level of ordinaryskill in the art, and suitable equipment and reagents are available fromcommercial suppliers. See generally Glick and Pasternak, MolecularBiotechnology, Principles & Applications of Recombinant DNA, ASM Press,Washington, D.C., 1994; Italcum et al., Ann. Rev. Biochem. 53:323-356,1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637, 1990.

In another aspect, materials and methods are provided for producing thepolypeptide fusions of the present invention, including dimeric proteinscomprising the fusion polypeptides. The fusion polypeptides can beproduced in genetically engineered host cells according to conventionaltechniques. Suitable host cells are those cell types that can betransformed or transfected with exogenous DNA and grown in culture, andinclude bacteria, fungal cells, and cultured higher eukaryotic cells(including cultured cells of multicellular organisms), particularlycultured mammalian cells. Techniques for manipulating cloned DNAmolecules and introducing exogenous DNA into a variety of host cells aredisclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, and Ausubel et al., eds., Current Protocols in Molecular Biology,Green and Wiley and Sons, N Y, 1993.

In general, a DNA sequence encoding a fusion polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct an ApoA-1 fusion polypeptide into the secretory pathway of ahost cell, a secretory signal sequence is provided in the expressionvector. The secretory signal sequence may be that of the native ApoA-1polypeptide, or may be derived from another secreted protein (e.g.,t-PA; see U.S. Pat. No. 5,641,655) or synthesized de novo. An engineeredcleavage site may be included at the junction between the secretorypeptide and the remainder of the polypeptide fusion to optimizeproteolytic processing in the host cell. The secretory signal sequenceis operably linked to the DNA sequence encoding the polypeptide fusion,i.e., the two sequences are joined in the correct reading frame andpositioned to direct the newly synthesized polypeptide fusion into thesecretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830). Secretory signalsequences suitable for use in accordance with the present inventioninclude, for example, polynucleotides encoding amino acid residues 1-18of SEQ ID NO:2.

Expression of fusion polypeptides comprising a dimerizing domain, via ahost cell secretory pathway, is expected to result in the production ofdimeric proteins. Accordingly, in another aspect, the present inventionprovides dimeric proteins comprising first and second fusionpolypeptides as described above (e.g., a dimeric protein comprising afirst fusion polypeptide and a second fusion polypeptide, where each ofthe first and second fusion polypeptides comprises, from anamino-terminal position to a carboxyl-terminal position, ApoA1-L1-D orApoA1-L1-D-L2-P as described herein). Dimers may also be assembled invitro upon incubation of component polypeptides under suitableconditions. In general, in vitro assembly will include incubating theprotein mixture under denaturing and reducing conditions followed byrefolding and reoxidation of the polypeptides to form dimers. Recoveryand assembly of proteins expressed in bacterial cells is disclosedbelow.

Cultured mammalian cells are suitable hosts for use within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., supra), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed by, for example, Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g., CHO-K1, ATCC No. CCL 61;CHO-DG44, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980)cell lines. Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Manassas, Va. Strong transcription promoters can be used,such as promoters from SV-40, cytomegalovirus, or myeloproliferativesarcoma virus. See, e.g., U.S. Pat. No. 4,956,288 and U.S. PatentApplication Publication No. 20030103986. Other suitable promotersinclude those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and4,601,978) and the adenovirus major late promoter. Expression vectorsfor use in mammalian cells include pZP-1, pZP-9, and pZMP21, which havebeen deposited with the American Type Culture Collection, 10801University Blvd., Manassas, Va. USA under accession numbers 98669,98668, and PTA-5266, respectively, and derivatives of these vectors.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants.” Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Anexemplary selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g., hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Cell-surface markers and otherphenotypic selection markers can be used to facilitate identification oftransfected cells (e.g., by fluorescence-activated cell sorting), andinclude, for example, CD8, CD4, nerve growth factor receptor, greenfluorescent protein, and the like.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPOpublication WO 94/06463.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See King and Possee, The Baculovirus Expression System: A LaboratoryGuide, Chapman & Hall, London; O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual, Oxford University Press., New York, 1994;and Richardson, Ed., Baculovirus Expression Protocols. Methods inMolecular Biology, Humana Press, Totowa, N.J., 1995. Recombinantbaculovirus can also be produced through the use of a transposon-basedsystem described by Luckow et al. (J. Virol. 67:4566-4579, 1993). Thissystem, which utilizes transfer vectors, is commercially available inkit form (BAC-TO-BAC kit; Life Technologies, Gaithersburg, Md.). Thetransfer vector (e.g., PFASTBAC1; Life Technologies) contains a Tn7transposon to move the DNA encoding the protein of interest into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk andRapoport, J. Biol. Chem. 270:1543-1549, 1995. Using techniques known inthe art, a transfer vector encoding a polypeptide fusion is transformedinto E. coli host cells, and the cells are screened for bacmids whichcontain an interrupted lacZ gene indicative of recombinant baculovirus.The bacmid DNA containing the recombinant baculovirus genome isisolated, using common techniques, and used to transfect Spodopterafrugiperda cells, such as Sf9 cells. Recombinant virus that expressesthe polypeptide fusion is subsequently produced. Recombinant viralstocks are made by methods commonly used the art.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HIGH FIVEcells; Invitrogen, Carlsbad, Calif.). See generally Glick and Pasternak,supra. See also U.S. Pat. No. 5,300,435. Serum-free media are used togrow and maintain the cells. Suitable media formulations are known inthe art and can be obtained from commercial suppliers. The cells aregrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells, at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3. Procedures used are generally described in available laboratorymanuals (e.g., King and Possee, supra; O'Reilly et al., supra.;Richardson, supra).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). An exemplary vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936; and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillernondii, and Candida maltosaare known in the art. See, e.g., Gleeson et al., J. Gen. Microbiol.132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; and Raymond et al.,Yeast 14:11-23, 1998. Aspergillus cells may be utilized according to themethods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533. Production of recombinant proteinsin Pichia methanolica is disclosed in U.S. Pat. Nos. 5,716,808;5,736,383; 5,854,039; and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well-known in theart (see, e.g., Sambrook et al., supra). When expressing a fusionpolypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine HCl or urea. The denatured polypeptide canthen be refolded and dimerized by diluting the denaturant, such as bydialysis against a solution of urea and a combination of reduced andoxidized glutathione, followed by dialysis against a buffered salinesolution. In the alternative, the protein may be recovered from thecytoplasm in soluble form and isolated without the use of denaturants.The protein is recovered from the cell as an aqueous extract in, forexample, phosphate buffered saline. To capture the protein of interest,the extract is applied directly to a chromatographic medium, such as animmobilized antibody or heparin-Sepharose column. Secreted polypeptidescan be recovered from the periplasmic space in a soluble and functionalform by disrupting the cells (by, for example, sonication or osmoticshock) and recovering the protein, thereby obviating the need fordenaturation and refolding. See, e.g., Lu et al., J. Immunol. Meth.267:213-226, 2002.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Proteins of the present invention are purified by conventional proteinpurification methods, typically by a combination of chromatographictechniques. See generally Affinity Chromatography: Principles & Methods,Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes, ProteinPurification: Principles and Practice, Springer-Verlag, New York, 1994.Proteins comprising an immunoglobulin heavy chain polypeptide can bepurified by affinity chromatography on immobilized protein A. Additionalpurification steps, such as gel filtration, can be used to obtain thedesired level of purity or to provide for desalting, buffer exchange,and the like.

For example, fractionation and/or conventional purification methods canbe used to obtain fusion polypeptides and dimeric proteins of thepresent invention purified from recombinant host cells. In general,ammonium sulfate precipitation and acid or chaotrope extraction may beused for fractionation of samples. Exemplary purification steps mayinclude hydroxyapatite, size exclusion, FPLC and reverse-phase highperformance liquid chromatography. Suitable chromatographic mediainclude derivatized dextrans, agarose, cellulose, polyacrylamide,specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives aresuitable. Exemplary chromatographic media include those mediaderivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media arewell-known and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, e.g., AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988);and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in protein isolation and purification can bedevised by those of skill in the art. For example, antibodies thatspecifically bind a fusion polypeptide or dimeric protein as describedherein (e.g., an antibody that specifically binds a polypeptide segmentcorresponding to ApoA-1) can be used to isolate large quantities ofprotein by immunoaffinity purification.

The proteins of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (see,e.g., M. Deutscher, (ed.), Meth. Enzymol. 182:529, 1990). Withinadditional embodiments of the invention, a fusion of the polypeptide ofinterest and an affinity tag (e.g., maltose-binding protein, animmunoglobulin domain) may be constructed to facilitate purification.Moreover, receptor- or ligand-binding properties of a fusion polypeptideor dimer thereof can be exploited for purification. For example, afusion polypeptide comprising an Aβ-binding polypeptide segment may beisolated by using affinity chromatography wherein amyloid beta (Aβ)peptide is bound to a column and the fusion polypeptide is bound andsubsequently eluted using standard chromatography methods.

The polypeptides of the present invention are typically purified to atleast about 80% purity, more typically to at least about 90% purity andpreferably to at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% purity with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. The polypeptides ofthe present invention may also be purified to a pharmaceutically purestate, which is greater than 99.9% pure. In certain preparations,purified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

IV. Methods of Use and Pharmaceutical Compositions

The fusion polypeptides and dimeric proteins of the present inventioncan be used to provide ApoA-1-mediated therapy for the treatment ofvarious diseases or disorders. In some aspects relating to bispecificfusions further comprising a second polypeptide segment as describedherein (e.g., an RNase, paraoxonase, platelet-activating factoracetylhydrolase (PAF-AH), cholesterol ester transfer protein (CETP), orlecithin-cholesterol acyltransferase (LCAT)), the fusion polypeptidesand dimeric proteins may further provide one or more additionalbiological activities for such treatment.

In particular aspects, the present invention provides methods fortreating a disease or disorder selected from a cardiovascular diseasecharacterized by atherosclerosis, a neurodegenerative disease, a diseasecharacterized by amyloid deposit, an autoimmune disease, an inflammatorydisease, an infectious disease, obesity, metabolic syndrome, nephroticsyndrome, burns, exposure to sulfur mustard gas, exposure to anorganophosphate, sepsis, and cancer. The methods generally includeadministering to a subject having the disease or disorder an effectiveamount of a fusion polypeptide or dimeric protein as described herein.

Atherosclerotic cardiovascular diseases amenable to treatment inaccordance with the present invention include, for example, coronaryheart disease and stroke. In some variations of treatment of coronaryheart disease, the coronary heart disease is characterized by acutecoronary syndrome. In some embodiments, the atheroscleroticcardiovascular disease is selected from cerebral artery disease (e.g.,extracranial cerebral artery disease, intracranial cerebral arterydisease), arteriosclerotic aortic disease, renal artery disease,mesenteric artery disease, and peripheral artery disease (e.g.,aortoiliac occlusive disease).

Neurodegenerative diseases amenable to treatment in accordance with thepresent invention include, for example, neurodegenerative diseasescharacterized by amyloid deposit and/or dementia. An exemplaryneurodegenerative disease characterized by amyloid deposit isAlzheimer's disease. Exemplary neurodegenerative diseases characterizedby dementia include Alzheimer's disease, Parkinson's disease,Huntington's disease, and amylotrophic lateral sclerosis (ALS). In someembodiments, the neurodegenerative disease is an inflammatory diseasesuch as, for example, a demyelinating inflammatory disease of the CNS(e.g., multiple sclerosis (MS), including, for example, spino-opticalMS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS)).

In some embodiments of a method for treating a neurodegenerative disease(e.g., Alzheimer's disease or Parkinson's disease), a fusion moleculefor the neurodegenerative disease treatment is a polypeptide having thestructure ApoA1-L1-D-L2-RNase (e.g., ApoA1-L1-[Fc region]-L2-RNase1) orApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fc region]-L2-PON1), or adimeric protein formed by dimerization of any of the foregoing fusionpolypeptides; in some such embodiments, the fusion polypeptide comprisesor consists of an amino acid sequence having at least 90%, at least 95%,or 100% identity with (i) amino acid residues 19-675 or 25-675 of SEQ IDNO:4, (ii) amino acid residues 19-657 or 25-675 of SEQ ID NO:14, (iii)amino acid residues 19-671 or 25-671 of SEQ ID NO:58, (iv) amino acidresidues 19-671 or 25-671 of SEQ ID NO:59; (v) amino acid residues19-883 or 25-883 of SEQ ID NO:28, (vi) amino acid residues 19-873 or25-873 of SEQ ID NO:38, (vii) amino acid residues 19-883 or 25-883 ofSEQ ID NO:46, or (viii) amino acid residues 19-883 or 25-883 of SEQ IDNO:48.

Autoimmune diseases amenable to treatment in accordance with the presentinvention include, for example, rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, and type 1 diabetes. In otherembodiments, the autoimmune disease is selected from coeliac disease,neuritis, polymyositis, juvenile rheumatoid arthritis, psoriasis,psoriatic arthritis, vitiligo, Sjogren's syndrome, autoimmunepancreatitis, an inflammatory bowel disease (e.g., Crohn's disease,ulcerative colitis), active chronic hepatitis, glomerulonephritis, lupusnephritis, scleroderma, antiphospholipid syndrome, autoimmunevasculitis, sarcoidosis, autoimmune thyroid diseases, Hashimoto'sthyroiditis, Graves disease, Wegener's granulomatosis, myastheniagravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris,primary biliary cirrhosis, pernicious anemia, sympathetic opthalmia,uveitis, autoimmune hemolytic anemia, pulmonary fibrosis, chronicberyllium disease, and idiopathic pulmonary fibrosis. In somevariations, the autoimmune disease is selected from rheumatoidarthritis, juvenile rheumatoid arthritis, psoriatic arthritis, systemiclupus erythematosus, lupus nephritis, scleroderma, psoriasis, Sjogren'ssyndrome, type 1 diabetes, antiphospholipid syndrome, and autoimmunevasculitis.

In some embodiments, a fusion molecule for treatment of an autoimmunedisease is a polypeptide having the structure ApoA1-L1-D (e.g.,ApoA1-L1-[Fc region]), ApoA1-L1-D-L2-RNase (e.g., ApoA1-L1-[Fcregion]-L2-RNase1), or ApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fcregion]-L2-PON1), or a dimeric protein formed by dimerization of any ofthe foregoing fusion polypeptides; in some such embodiments, the fusionpolypeptide comprises or consists of an amino acid sequence having atleast 90%, at least 95%, or 100% identity with (i) amino acid residues19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii) amino acidresidues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13, (iii) aminoacid residues 19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20, (iv)amino acid residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22,(v) amino acid residues 19-535, 19-534, 25-535, or 25-534 of SEQ IDNO:24, (vi) amino acid residues 19-675 or 25-675 of SEQ ID NO:4, (vii)amino acid residues 19-657 or 25-675 of SEQ ID NO:14, (viii) amino acidresidues 19-671 or 25-671 of SEQ ID NO:58, (ix) amino acid residues19-671 or 25-671 of SEQ ID NO:59; (x) amino acid residues 19-883 or25-883 of SEQ ID NO:28, (xi) amino acid residues 19-873 or 25-873 of SEQID NO:38, (xii) amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or(xiii) amino acid residues 19-883 or 25-883 of SEQ ID NO:48. In someparticular variations of a method for treating rheumatoid arthritis(RA), a fusion molecule for the RA treatment is a polypeptide having thestructure ApoA1-L1-D (e.g., ApoA1-L1-[Fc region]), or a dimeric proteinformed by dimerization of the foregoing fusion polypeptide; in some suchembodiments, the fusion polypeptide comprises or consists of an aminoacid sequence having at least 90%, at least 95%, or 100% identity with(i) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:2, (ii) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:13, (iii) amino acid residues 19-501, 19-500, 25-501, or 25-500 ofSEQ ID NO:20, (iv) amino acid residues 19-515, 19-514, 25-515, or 25-514of SEQ ID NO:22, or (v) amino acid residues 19-535, 19-534, 25-535, or25-534 of SEQ ID NO:24. In some particular variations of a method fortreating systemic lupus erythematosus (SLE), a fusion molecule for theSLE treatment is a polypeptide having the structure ApoA1-L1-D-L2-RNase(e.g., ApoA1-L1-[Fc region]-L2-RNase1) or ApoA1-L1-D-L2-paraoxonase(e.g., ApoA1-L1-[Fc region]-L2-PON1), or a dimeric protein formed bydimerization of any of the foregoing fusion polypeptides; in some suchembodiments, the fusion polypeptide comprises or consists of an aminoacid sequence having at least 90%, at least 95%, or 100% identity with(i) amino acid residues 19-675 or 25-675 of SEQ ID NO:4, (ii) amino acidresidues 19-657 or 25-675 of SEQ ID NO:14, (iii) amino acid residues19-671 or 25-671 of SEQ ID NO:58, (iv) amino acid residues 19-671 or25-671 of SEQ ID NO:59, (v) amino acid residues 19-883 or 25-883 of SEQID NO:28, (vi) amino acid residues 19-873 or 25-873 of SEQ ID NO:38,(vii) amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or (viii)amino acid residues 19-883 or 25-883 of SEQ ID NO:48. In some particularvariations of a method for treating multiple sclerosis (MS), a fusionmolecule for the MS treatment is a polypeptide having the structureApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fc region]-L2-PON1), or adimeric protein formed by dimerization of the foregoing fusionpolypeptide; in some such embodiments, the fusion polypeptide comprisesor consists of an amino acid sequence having at least 90%, at least 95%,or 100% identity with (i) amino acid residues 19-883 or 25-883 of SEQ IDNO:28, (ii) amino acid residues 19-873 or 25-873 of SEQ ID NO:38, (iii)amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) amino acidresidues 19-883 or 25-883 of SEQ ID NO:48.

Inflammatory diseases amenable to treatment in accordance with thepresent invention include, for example, rheumatoid arthritis, systemiclupus erythematosus, multiple sclerosis, type 1 diabetes, type 2diabetes, and obesity. In some embodiments, the inflammatory disease isan neurodegenerative inflammatory disease such as, for example, multiplesclerosis, Alzheimer's disease, or Parkinson's disease. In otherembodiments, the inflammatory disease is an atherosclerotic disease(e.g., coronary heart disease or stroke). In yet other variations, theinflammatory disease is selected from hepatitis (e.g., non-alcoholicsteatohepatitis), ankylosing spondylitis, arthritis (e.g.,osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), Crohn'sdisease, ulcerative colitis, dermatitis, diverticulitis, fibromyalgia,irritable bowel syndrome (IBS), and nephritis. In other embodiments, theinflammatory disease is an inflammatory lung disease; in some suchembodiments, the inflammatory lung disease is selected from asthma,chronic obstructive pulmonary disease (COPD), bronchiectasis, idiopathicpulmonary fibrosis, hyperoxia, hypoxia, and acute respiratory distresssyndrome (ARDS). In some variations, a patient having the inflammatorylung disease is a patient that has been exposed to sulfur mustard gas(SM). In other variations, a patient having the inflammatory lungdisease is a patient that has been exposed to an organophosphate, suchas an insecticide or other neurotoxin.

In some embodiments, a fusion molecule for treatment of an inflammatorydisease (e.g., an inflammatory lung disease) is a polypeptide having thestructure ApoA1-L1-D (e.g., ApoA1-L1-[Fc region]), ApoA1-L1-D-L2-RNase(e.g., ApoA1-L1-[Fc region]-L2-RNase1), or ApoA1-L1-D-L2-paraoxonase(e.g., ApoA1-L1-[Fc region]-L2-PON1), or a dimeric protein formed bydimerization of any of the foregoing fusion polypeptides; in some suchembodiments, the fusion polypeptide comprises or consists of an aminoacid sequence having at least 90%, at least 95%, or 100% identity with(i) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:2, (ii) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:13, (iii) amino acid residues 19-501, 19-500, 25-501, or 25-500 ofSEQ ID NO:20, (iv) amino acid residues 19-515, 19-514, 25-515, or 25-514of SEQ ID NO:22, (v) amino acid residues 19-535, 19-534, 25-535, or25-534 of SEQ ID NO:24, (vi) amino acid residues 19-675 or 25-675 of SEQID NO:4, (vii) amino acid residues 19-657 or 25-675 of SEQ ID NO:14,(viii) amino acid residues 19-671 or 25-671 of SEQ ID NO:58, (ix) aminoacid residues 19-671 or 25-671 of SEQ ID NO:59; (x) amino acid residues19-883 or 25-883 of SEQ ID NO:28, (xi) amino acid residues 19-873 or25-873 of SEQ ID NO:38, (xii) amino acid residues 19-883 or 25-883 ofSEQ ID NO:46, or (xiii) amino acid residues 19-883 or 25-883 of SEQ IDNO:48. In some particular variations of a method for treating idiopathicpulmonary fibrosis, a fusion molecule for the idiopathic pulmonaryfibrosis treatment is a polypeptide having the structure ApoA1-L1-D(e.g., ApoA1-L1-[Fc region]), or a dimeric protein formed bydimerization of the foregoing fusion polypeptide; in some suchembodiments, the fusion polypeptide comprises or consists of an aminoacid sequence having at least 90%, at least 95%, or 100% identity with(i) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:2, (ii) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:13, (iii) amino acid residues 19-501, 19-500, 25-501, or 25-500 ofSEQ ID NO:20, (iv) amino acid residues 19-515, 19-514, 25-515, or 25-514of SEQ ID NO:22, or (v) amino acid residues 19-535, 19-534, 25-535, or25-534 of SEQ ID NO:24. In some particular variations of a method fortreating an inflammatory lung disease in a patient that has been exposedto sulfur mustard gas (SM) or an organophosphate, a fusion molecule forthe treatment is a polypeptide having the structureApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fc region]-L2-PON1), or adimeric protein formed by dimerization of the foregoing fusionpolypeptide; in some such embodiments, the fusion polypeptide comprisesor consists of an amino acid sequence having at least 90%, at least 95%,or 100% identity with (i) amino acid residues 19-883 or 25-883 of SEQ IDNO:28, (ii) amino acid residues 19-873 or 25-873 of SEQ ID NO:38, (iii)amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) amino acidresidues 19-883 or 25-883 of SEQ ID NO:48. In some particular variationsof a method for treating acute respiratory distress syndrome (ARDS),hypoxia, or hyperoxia, a fusion molecule for the treatment is apolypeptide having the structure ApoA1-L1-D-L2-RNase (e.g., ApoA1-L1-[Fcregion]-L2-RNase1), or a dimeric protein formed by dimerization of anyof the foregoing fusion polypeptides; in some such embodiments, thefusion polypeptide comprises or consists of an amino acid sequencehaving at least 90%, at least 95%, or 100% identity with (i) amino acidresidues 19-675 or 25-675 of SEQ ID NO:4, (ii) amino acid residues19-657 or 25-675 of SEQ ID NO:14, (iii) amino acid residues 19-671 or25-671 of SEQ ID NO:58, or (iv) amino acid residues 19-671 or 25-671 ofSEQ ID NO:59. In certain embodiments, such ApoA1-L1-D-L2-RNasevariations are used to treat premature infants that are treated withoxygen for an extended period of time.

In some embodiments, a fusion molecule for treatment of exposure tosulfur mustard gas (SM) or exposure to an organophosphate is apolypeptide having the structure ApoA1-L1-D-L2-paraoxonase (e.g.,ApoA1-L1-[Fc region]-L2-PON1), or a dimeric protein formed bydimerization of the foregoing fusion polypeptide; in some suchembodiments, the fusion polypeptide comprises or consists of an aminoacid sequence having at least 90%, at least 95%, or 100% identity with(i) amino acid residues 19-883 or 25-883 of SEQ ID NO:28, (ii) aminoacid residues 19-873 or 25-873 of SEQ ID NO:38, (iii) amino acidresidues 19-883 or 25-883 of SEQ ID NO:46, or (iv) amino acid residues19-883 or 25-883 of SEQ ID NO:48.

Infectious diseases amenable to treatment in accordance with the presentinvention include, for example, bacterial infections and parasiticinfections. In some embodiments, the parasitic infection is aTrypanosoma brucei or Leishmania infection. In other embodiments, thebacterial infection is a Pseudomonas aeruginosa infection.

In some embodiments of a method for treating a Pseudomonas aeruginosainfection, a fusion molecule for the Pseudomonas aeruginosa infectiontreatment is a polypeptide having the structureApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fc region]-L2-PON1), or adimeric protein formed by dimerization of the foregoing fusionpolypeptide; in some such embodiments, the fusion polypeptide comprisesor consists of an amino acid sequence having at least 90%, at least 95%,or 100% identity with (i) amino acid residues 19-883 or 25-883 of SEQ IDNO:28, (ii) amino acid residues 19-873 or 25-873 of SEQ ID NO:38, (iii)amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) amino acidresidues 19-883 or 25-883 of SEQ ID NO:48.

In some embodiments, a fusion molecule for treatment of an infectiousdisease (e.g., an inflammatory lung disease) is a polypeptide having thestructure ApoA1-L1-D (e.g., ApoA1-L1-[Fc region]), or a dimeric proteinformed by dimerization of the foregoing fusion polypeptide; in some suchembodiments, the fusion polypeptide comprises or consists of an aminoacid sequence having at least 90%, at least 95%, or 100% identity with(i) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQ IDNO:2, (ii) amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQID NO:13, (iii) amino acid residues 19-501, 19-500, 25-501, or 25-500 ofSEQ ID NO:20, (iv) amino acid residues 19-515, 19-514, 25-515, or 25-514of SEQ ID NO:22, or (v) amino acid residues 19-535, 19-534, 25-535, or25-534 of SEQ ID NO:24.

In some embodiments, a fusion molecule for treatment of sepsis is apolypeptide having the structure ApoA1-L1-D (e.g., ApoA1-L1-[Fc region])or ApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fc region]-L2-PON1), or adimeric protein formed by dimerization of any of the foregoing fusionpolypeptides; in some such embodiments, the fusion polypeptide comprisesor consists of an amino acid sequence having at least 90%, at least 95%,or 100% identity with (i) amino acid residues 19-525, 19-524, 25-525, or25-524 of SEQ ID NO:2, (ii) amino acid residues 19-525, 19-524, 25-525,or 25-524 of SEQ ID NO:13, (iii) amino acid residues 19-501, 19-500,25-501, or 25-500 of SEQ ID NO:20, (iv) amino acid residues 19-515,19-514, 25-515, or 25-514 of SEQ ID NO:22, (v) amino acid residues19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24, (vi) amino acidresidues 19-883 or 25-883 of SEQ ID NO:28, (vii) amino acid residues19-873 or 25-873 of SEQ ID NO:38, (viii) amino acid residues 19-883 or25-883 of SEQ ID NO:46, or (ix) amino acid residues 19-883 or 25-883 ofSEQ ID NO:48.

Cancers that may be treated in accordance with the present inventioninclude, for example, the following: a cancer of the head and neck(e.g., a cancer of the oral cavity, orophyarynx, nasopharynx,hypopharynx, nasal cavity or paranasal sinuses, larynx, lip, or salivarygland); a lung cancer (e.g., non-small cell lung cancer, small cellcarcinoma, or mesothelimia); a gastrointestinal tract cancer (e.g.,colorectal cancer, gastric cancer, esophageal cancer, or anal cancer);gastrointestinal stromal tumor (GIST); pancreatic adenocarcinoma;pancreatic acinar cell carcinoma; a cancer of the small intestine; acancer of the liver or biliary tree (e.g., liver cell adenoma,hepatocellular carcinoma, hemangiosarcoma, extrahepatic or intrahepaticcholangiosarcoma, cancer of the ampulla of vater, or gallbladdercancer); a breast cancer (e.g., metastatic breast cancer or inflammatorybreast cancer); a gynecologic cancer (e.g., cervical cancer, ovariancancer, fallopian tube cancer, peritoneal carcinoma, vaginal cancer,vulvar cancer, gestational trophoblastic neoplasia, or uterine cancer,including endometrial cancer or uterine sarcoma); a cancer of theurinary tract (e.g., prostate cancer; bladder cancer; penile cancer;urethral cancer, or kidney cancer such as, for example, renal cellcarcinoma or transitional cell carcinoma, including renal pelvis andureter); testicular cancer; a cancer of the central nervous system (CNS)such as an intracranial tumor (e.g., astrocytoma, anaplasticastrocytoma, glioblastoma, oligodendroglioma, anaplasticoligodendroglioma, ependymoma, primary CNS lymphoma, medulloblastoma,germ cell tumor, pineal gland neoplasm, meningioma, pituitary tumor,tumor of the nerve sheath (e.g., schwannoma), chordoma,craniopharyngioma, a chloroid plexus tumor (e.g., chloroid plexuscarcinoma); or other intracranial tumor of neuronal or glial origin) ora tumor of the spinal cord (e.g., schwannoma, meningioma); an endocrineneoplasm (e.g., thyroid cancer such as, for example, thyroid carcinoma,medullary cancer, or thyroid lymphoma; a pancreatic endocrine tumor suchas, for example, an insulinoma or glucagonoma; an adrenal carcinoma suchas, for example, pheochromocytoma; a carcinoid tumor; or a parathyroidcarcinoma); a skin cancer (e.g., squamous cell carcinoma; basal cellcarcinoma; Kaposi's sarcoma; or a malignant melanoma such as, forexample, an intraocular melanoma); a bone cancer (e.g., a bone sarcomasuch as, for example, osteosarcoma, osteochondroma, or Ewing's sarcoma);multiple myeloma; a chloroma; a soft tissue sarcoma (e.g., a fibroustumor or fibrohistiocytic tumor); a tumor of the smooth muscle orskeletal muscle; a blood or lymph vessel perivascular tumor (e.g.,Kaposi's sarcoma); a synovial tumor; a mesothelial tumor; a neuraltumor; a paraganglionic tumor; an extraskeletal cartilaginous or osseoustumor; and a pluripotential mesenchymal tumor. In some such embodiments,an ApoA-1 fusion molecule as described herein is administered to acancer patient as one of the distinct therapies of a combination therapysuch as, for example, a combination therapy comprising anon-ApoA1-mediated immunomodulatory therapy (e.g., a therapy comprisingan immune checkpoint inhibitor), a radiation therapy, or a chemotherapy.

In certain embodiments, a combination cancer therapy comprises an ApoA-1fusion molecule as described herein and a targeted therapy such as,e.g., a therapeutic monoclonal antibody targeting a specificcell-surface or extracellular antigen, or a small molecule targeting anintracellular protein (e.g., an intracellular enzyme). Exemplaryantibody targeted therapies include anti-VEGF (e.g., bevacizumab),anti-EGFR (e.g., cetuximab), anti-CTLA-4 (e.g., ipilimumab), anti-PD-1(e.g., nivolumab), and anti-PD-L1 (e.g., pembrolizumab). Exemplary smallmolecule targeted therapies include proteasome inhibitors (e.g.,bortezomib), tyrosine kinase inhibitors (e.g., imatinib),cyclin-dependent kinase inhibitors (e.g., seliciclib); BRAF inhibitors(e.g., vemurafenib or dabrafenib); and MEK kinase inhibitors (e.g.,trametnib).

In some cancer combination therapy variations comprising an immunecheckpoint inhibitor, the combination therapy includes ananti-PD-1/PD-L1 therapy, an anti-CTLA-4 therapy, or both. In certainaspects, ApoA-1 fusion molecules as described herein can increase theresponse rate to either anti-CTLA-4 or anti-PD-1/PD-L1 therapy, as wellas the response rate to the combination of anti-CTLA-4 plusanti-PD-1/PD-L1 therapy. Fusion molecules of the invention may also beuseful for reducing the toxicity associated with anti-CTLA-4,anti-PD-1/PD-L1, or the combination thereof.

In certain variations, a cancer treated in accordance with the presentinvention is selected from malignant melanoma, renal cell carcinoma,non-small cell lung cancer, bladder cancer, and head and neck cancer.These cancers have shown responses to immune checkpoint inhibitorsanti-PD-1/PD-L1 and anti-CTLA-4. See Grimaldi et al., Expert Opin. Biol.Ther. 16:433-41, 2016; Gunturi et al., Curr. Treat. Options Oncol.15:137-46, 2014; Topalian et al., Nat. Rev. Cancer 16:275-87, 2016.Thus, in some more specific variations, any of these cancers is treatedwith an ApoA-1 fusion molecule as described herein in combination withan anti-PD-1/PD-L1 therapy, an anti-CTLA-4 therapy, or both.

In some embodiments, a fusion molecule for treatment of a cancer is apolypeptide having the structure ApoA1-L1-D (e.g., ApoA1-L1-[Fcregion]), ApoA1-L1-D-L2-RNase (e.g., ApoA1-L1-[Fc region]-L2-RNase1), orApoA1-L1-D-L2-paraoxonase (e.g., ApoA1-L1-[Fc region]-L2-PON1), or adimeric protein formed by dimerization of any of the foregoing fusionpolypeptides; in some such embodiments, the fusion polypeptide comprisesor consists of an amino acid sequence having at least 90%, at least 95%,or 100% identity with (i) amino acid residues 19-525, 19-524, 25-525, or25-524 of SEQ ID NO:2, (ii) amino acid residues 19-525, 19-524, 25-525,or 25-524 of SEQ ID NO:13, (iii) amino acid residues 19-501, 19-500,25-501, or 25-500 of SEQ ID NO:20, (iv) amino acid residues 19-515,19-514, 25-515, or 25-514 of SEQ ID NO:22, (v) amino acid residues19-535, 19-534, 25-535, or 25-534 of SEQ ID NO:24, (vi) amino acidresidues 19-675 or 25-675 of SEQ ID NO:4, (vii) amino acid residues19-657 or 25-675 of SEQ ID NO:14, (viii) amino acid residues 19-671 or25-671 of SEQ ID NO:58, (ix) amino acid residues 19-671 or 25-671 of SEQID NO:59; (x) amino acid residues 19-883 or 25-883 of SEQ ID NO:28, (xi)amino acid residues 19-873 or 25-873 of SEQ ID NO:38, (xii) amino acidresidues 19-883 or 25-883 of SEQ ID NO:46, or (xiii) amino acid residues19-883 or 25-883 of SEQ ID NO:48.

In certain embodiments for treatment of a disease or disorder asdescribed herein, the fusion polypeptide comprises an amino acidsequence having at least 90% or at least 95% identity with (i) aminoacid residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii)amino acid residues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13,(iii) amino acid residues 19-501, 19-500, 25-501, or 25-500 of SEQ IDNO:20, (iv) amino acid residues 19-515, 19-514, 25-515, or 25-514 of SEQID NO:22, (v) amino acid residues 19-535, 19-534, 25-535, or 25-534 ofSEQ ID NO:24, (vi) amino acid residues 19-675 or 25-675 of SEQ ID NO:4,(vii) amino acid residues 19-657 or 25-675 of SEQ ID NO:14, (viii) aminoacid residues 19-671 or 25-671 of SEQ ID NO:58, (ix) amino acid residues19-671 or 25-671 of SEQ ID NO:59; (x) amino acid residues 19-883 or25-883 of SEQ ID NO:28, (xi) amino acid residues 19-873 or 25-873 of SEQID NO:38, (xii) amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or(xiii) amino acid residues 19-883 or 25-883 of SEQ ID NO:48, where thefusion polypeptide comprises at least one amino acid substitution in thefirst (“ApoA1”) polypeptide segment selected from V156[E/K],Y192[S/Q/N/H/F], M86[L/I/V], M112[L/I/V], M148[L/I/V], W8F, W50F, W72F,and W132F as described herein. In some treatment embodiments where thefusion polypeptide has the structure ApoA1-L1-D-L2-paraoxonase (e.g.,ApoA1-L1-[Fc region]-L2-PON1), the fusion polypeptide comprises an aminoacid sequence having at least 90% or at least 95% identity with (i)amino acid residues 19-883 or 25-883 of SEQ ID NO:28, (ii) amino acidresidues 19-873 or 25-873 of SEQ ID NO:38, (iii) amino acid residues19-883 or 25-883 of SEQ ID NO:46, or (iv) amino acid residues 19-883 or25-883 of SEQ ID NO:48, where the fusion polypeptide comprises asubstitution in the second (“P”) polypeptide segment selected fromY185[H/Q/S] and F293[H/Q/N] as described herein; in some suchvariations, the fusion polypeptide comprises at least one of theforegoing substitutions in the ApoA1 segment. In some treatmentembodiments where the fusion polypeptide comprises one or moresubstitutions as above, the fusion polypeptide comprises an amino acidsequence that is otherwise 100% identical to (i) amino acid residues19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:2, (ii) amino acidresidues 19-525, 19-524, 25-525, or 25-524 of SEQ ID NO:13, (iii) aminoacid residues 19-501, 19-500, 25-501, or 25-500 of SEQ ID NO:20, (iv)amino acid residues 19-515, 19-514, 25-515, or 25-514 of SEQ ID NO:22,(v) amino acid residues 19-535, 19-534, 25-535, or 25-534 of SEQ IDNO:24, (vi) amino acid residues 19-675 or 25-675 of SEQ ID NO:4, (vii)amino acid residues 19-657 or 25-675 of SEQ ID NO:14, (viii) amino acidresidues 19-671 or 25-671 of SEQ ID NO:58, (ix) amino acid residues19-671 or 25-671 of SEQ ID NO:59; (x) amino acid residues 19-883 or25-883 of SEQ ID NO:28, (xi) amino acid residues 19-873 or 25-873 of SEQID NO:38, (xii) amino acid residues 19-883 or 25-883 of SEQ ID NO:46, or(xiii) amino acid residues 19-883 or 25-883 of SEQ ID NO:48.

In some embodiments, an ApoA-1 fusion molecule as described herein isadministered to a patient as one of the distinct therapies of acombination therapy comprising administration of a myeloperoxidase (MPO)inhibitor. MPO can oxidize and inactivate ApoA-1 and PON1, so inhibitionof MPO during the period of therapy can protect the fusion molecules ofthe present invention, including, for example, bispecific fusionmolecules further comprising a paraoxonase (e.g., PON1). After therapywith fusion molecules of the present invention, production of MPO isexpected to be suppressed, and the treated individual is expected tohave recovered redox balance and the ability to regulate MPO production.Because of the importance of myeloperoxidase in killing off bacteria byleukocytes, a combined short term use of MPO inhibitors and fusionmolecules of the present invention is preferred over a combined longterm use. Inhibitors of MPO are generally known in the art and include,for example, PF-06282999 (see Ruggeri et al., J. Med. Chem.58:8513-8528, 2015) and INV-315 (see Liu et al., PLoS ONE 7:e50767,2012).

For therapeutic use, a fusion polypeptide or dimeric protein asdescribed herein is delivered in a manner consistent with conventionalmethodologies associated with management of the disease or disorder forwhich treatment is sought. In accordance with the disclosure herein, aneffective amount of the fusion polypeptide or dimeric protein isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent or treat the disease or disorder.

Subjects for administration of fusion polypeptides or dimeric proteinsas described herein include patients at high risk for developing aparticular disease or disorder as well as patients presenting with anexisting disease or disorder. In certain embodiments, the subject hasbeen diagnosed as having the disease or disorder for which treatment issought. Further, subjects can be monitored during the course oftreatment for any change in the disease or disorder (e.g., for anincrease or decrease in clinical symptoms of the disease or disorder).Also, in some variations, the subject does not suffer from anotherdisease or disorder requiring treatment that involves administration ofan ApoA-1 protein.

In prophylactic applications, pharmaceutical compositions or medicantsare administered to a patient susceptible to, or otherwise at risk of, aparticular disease in an amount sufficient to eliminate or reduce therisk or delay the onset of the disease. In therapeutic applications,compositions or medicants are administered to a patient suspected of, oralready suffering from such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease and itscomplications. An amount adequate to accomplish this is referred to as atherapeutically or pharmaceutically effective dose or amount. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient response (e.g., atherosclerosisregression or stabilization of existing plaques in coronary heartdisease) has been achieved. Typically, the response is monitored andrepeated dosages are given if the desired response starts to fade.

To identify subject patients for treatment according to the methods ofthe invention, accepted screening methods may be employed to determinerisk factors associated with a specific disease or to determine thestatus of an existing disease identified in a subject. Such methods caninclude, for example, determining whether an individual has relativeswho have been diagnosed with a particular disease. Screening methods canalso include, for example, conventional work-ups to determine familialstatus for a particular disease known to have a heritable component.Toward this end, nucleotide probes can be routinely employed to identifyindividuals carrying genetic markers associated with a particulardisease of interest. In addition, a wide variety of immunologicalmethods are known in the art that are useful to identify markers forspecific diseases. Screening may be implemented as indicated by knownpatient symptomology, age factors, related risk factors, etc. Thesemethods allow the clinician to routinely select patients in need of themethods described herein for treatment. In accordance with thesemethods, treatment using a fusion polypeptide or dimeric protein of thepresent invention may be implemented as an independent treatment programor as a follow-up, adjunct, or coordinate treatment regimen to othertreatments.

For administration, a fusion polypeptide or dimeric protein inaccordance with the present invention is formulated as a pharmaceuticalcomposition. A pharmaceutical composition comprising a fusionpolypeptide or dimeric protein as described herein can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic molecule is combined in a mixturewith a pharmaceutically acceptable carrier. A composition is said to bea “pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, e.g., Gennaro (ed.),Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed.1995). Formulations may further include one or more excipients,preservatives, solubilizers, buffering agents, albumin to preventprotein loss on vial surfaces, etc.

A pharmaceutical composition comprising a fusion polypeptide or dimericprotein of the present invention is administered to a subject in aneffective amount. The fusion polypeptide or dimeric protein may beadministered to subjects by a variety of administration modes,including, for example, by intramuscular, subcutaneous, intravenous,intra-atrial, intra-articular, parenteral, intranasal, intrapulmonary,transdermal, intrapleural, intrathecal, and oral routes ofadministration. For prevention and treatment purposes, the fusionpolypeptide or dimeric protein may be administered to a subject in asingle bolus delivery, via continuous delivery (e.g., continuoustransdermal delivery) over an extended time period, or in a repeatedadministration protocol (e.g., on an hourly, daily, or weekly basis).

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby determining effective dosages and administration protocols thatsignificantly reduce the occurrence or severity of the subject diseaseor disorder in model subjects. Effective doses of the compositions ofthe present invention vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, whether treatment is prophylactic ortherapeutic, as well as the specific activity of the composition itselfand its ability to elicit the desired response in the individual.Usually, the patient is a human, but in some diseases, the patient canbe a nonhuman mammal. Typically, dosage regimens are adjusted to providean optimum therapeutic response, i.e., to optimize safety and efficacy.Accordingly, a therapeutically or prophylactically effective amount isalso one in which any undesired collateral effects are outweighed bybeneficial effects (e.g., in the case of treatment of atheroscleroticcardiovascular disease, where any undesired collateral effects areoutweighed by any beneficial effects such as increase in HDL,atherosclerosis regression, and/or plaque stabilization). Foradministration of a fusion polypeptide or dimeric protein of the presentinvention, a dosage typically ranges from about 0.1 μg to 100 mg/kg or 1μg/kg to about 50 mg/kg, and more usually 10 μg to 5 mg/kg of thesubject's body weight. In more specific embodiments, an effective amountof the agent is between about 1 μg/kg and about 20 mg/kg, between about10 μg/kg and about 10 mg/kg, or between about 0.1 mg/kg and about 5mg/kg. Dosages within this range can be achieved by single or multipleadministrations, including, e.g., multiple administrations per day ordaily, weekly, bi-weekly, or monthly administrations. For example, incertain variations, a regimen consists of an initial administrationfollowed by multiple, subsequent administrations at weekly or bi-weeklyintervals. Another regimen consists of an initial administrationfollowed by multiple, subsequent administrations at monthly orbi-monthly intervals. Alternatively, administrations can be on anirregular basis as indicated by monitoring of clinical symptoms of thedisease or disorder and/or monitoring of disease biomarkers or otherdisease correlates (e.g., HDL levels in the case of atheroscleroticcardiovascular disease).

Particularly suitable animal models for evaluating efficacy of an ApoA-1composition of the present invention for treatment of atherosclerosisinclude, for example, known mouse models that are deficient in the lowdensity lipoprotein receptor (LDLR) or ApoE. LDLR deficient mice developatherosclerotic plaques after eating a high fat diet for 12 weeks, andhuman ApoA-1 (reconstituted with lipids) is effective in reducingplaques in this model. ApoE deficient mice are also commonly used tostudy atherosclerosis, and human ApoA-1 (reconstituted with lipids)works rapidly in this model. Rabbits that are transgenic for hepaticlipase are another known atherosclerosis model for testing ApoA-1compositions.

One model of Alzheimer's disease uses overexpression of mutant amyloid-βprecursor protein (APP) and presenilin 1 in mice. In these mice,overexpression of human ApoA-1 prevented memory and learning deficits.See Lewis et al., J Biol. Chem. 285: 36958-36968, 2010.

Also known is the collagen-induced arthritis (CIA) model for rheumatoidarthritis (RA). CIA shares similar immunological and pathologicalfeatures with RA, making it an ideal model for evaluating efficacy ofApoA-1 compositions. See, e.g., Charles-Schoeman et al., Clin Immunol.127:234-44, 2008 (describing studies showing efficacy of the ApoA-1mimetic peptide, D-4F, in the CIA model). Another known model for RA isPG-polysaccharide (PG-PS)-induced arthritis in female Lewis rats. Inthese mice, administration of ApoA-1 protein or reconstituted HDLsreduced acute and chronic joint inflammation. Wu et al., ArteriosclerThromb Vasc Biol 34:543-551, 2014.

Animal models for multiple sclerosis (MS) include, for example,experimental allergic encephalomyelitis (EAE) models that rely on theinduction of an autoimmune response in the CNS by immunization with aCNS antigen (also referred to as an “encephalitogen” in the context ofEAE), which leads to inflammation, demyelination, and weakness. ApoA-1deficient mice have been shown to exhibit more neurodegeneration andworse disease than wild-type animals in this model. See Meyers et al.,J. Neuroimmunol. 277: 176-185, 2014.

Fusion molecules of the present invention can be evaluated foranti-tumor activity in animal tumor models such as, e.g., B16 melanoma,a poorly immunogenic tumor. Multiple models of tumor immunotherapy havebeen studied. See Ngiow et al., Adv. Immunol. 130:1-24, 2016. The B16melanoma model has been studied extensively with checkpoint inhibitorsanti-CTLA-4, anti-PD-1, and the combination thereof. Anti-CTLA-4 alonehas a potent therapeutic effect in this model only when combined withGM-CSF transduced tumor vaccine, or combined with anti-PD-1. See Weber,Semin. Oncol. 37:430-439, 2010; Ai et al., Cancer Immunol. Immunother.64:885-92, 2015; Haanen et al., Prog. Tumor Res. 42:55-66, 2015.Efficacy of an ApoA-1 fusion molecule for treatment of malignantmelanoma is shown, for example, by slowed tumor growth followingadministration to B16 melanoma mice that have formed palpablesubcutaneous tumor nodules. Efficacy of an ApoA-1 fusion molecule can beevaluated in B16 melanoma mice either alone or, alternatively, incombination with another anti-cancer therapy (e.g., anti-CTLA-4, with orwithout tumor vaccine or with or without anti-PD-1/PD-L1). For example,tumor rejection in B16 melanoma mice using a combination of an ApoA-1fusion molecule as described herein and anti-CTLA-4, in the absence oftumor vaccine, demonstrates an enhanced response to anti-CTLA-4 usingthe ApoA-1 therapy. In exemplary studies to evaluate ApoA-1 fusionmolecules comprising human protein sequences, which are functionallyactive in mice but are expected to be immunogenic in these models (andthereby likely to result in formation of neutralizing antibodies after7-10 days), mice may be administered a fusion molecule of the presentinvention for a short period (for example, one week, administered in,e.g., two doses of about 40 mg/kg three days apart), and tumor growththen monitored, typically for two to three weeks after injection withthe fusion molecule.

Dosage of the pharmaceutical composition may be varied by the attendingclinician to maintain a desired concentration at a target site. Forexample, if an intravenous mode of delivery is selected, localconcentration of the agent in the bloodstream at the target tissue maybe between about 1-50 nanomoles of the composition per liter, sometimesbetween about 1.0 nanomole per liter and 10, 15, or 25 nanomoles perliter depending on the subject's status and projected measured response.Higher or lower concentrations may be selected based on the mode ofdelivery, e.g., trans-epidermal delivery versus delivery to a mucosalsurface. Dosage should also be adjusted based on the release rate of theadministered formulation, e.g., nasal spray versus powder, sustainedrelease oral or injected particles, transdermal formulations, etc. Toachieve the same serum concentration level, for example, slow-releaseparticles with a release rate of 5 nanomolar (under standard conditions)would be administered at about twice the dosage of particles with arelease rate of 10 nanomolar.

A pharmaceutical composition comprising a fusion polypeptide or dimericprotein as described herein can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions, aerosols, droplets, topological solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants. See, e.g., Bremer et al., Pharm. Biotechnol. 10:239,1997; Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems95-123 (Ranade and Hollinger, eds., CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems 239-254 (Sanders and Hendren, eds., Plenum Press 1997); Yewey etal., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems 93-117 (Sanders andHendren, eds., Plenum Press 1997). Other solid forms include creams,pastes, other topological applications, and the like.

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer. See, e.g.,Gombotz and Pettit, Bioconjugate Chem. 6:332, 1995; Ranade, “Role ofPolymers in Drug Delivery,” in Drug Delivery Systems 51-93 (Ranade andHollinger, eds., CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems 45-92 (Sanders and Hendren, eds., PlenumPress 1997); Bartus et al., Science 281:1161, 1998; Putney and Burke,Nature Biotechnology 16:153, 1998; Putney, Curr. Opin. Chem. Biol.2:548, 1998. Polyethylene glycol (PEG)-coated nanospheres can alsoprovide carriers for intravenous administration of therapeutic proteins.See, e.g., Gref et al., Pharm. Biotechnol. 10:167, 1997.

Other dosage forms can be devised by those skilled in the art, as shownby, e.g., Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems (Lea & Febiger, 5th ed. 1990); Gennaro (ed.),Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed.1995), and Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).

Pharmaceutical compositions as described herein may also be used in thecontext of combination therapy. The term “combination therapy” is usedherein to denote that a subject is administered at least onetherapeutically effective dose of a fusion polypeptide or dimericprotein as described herein and another therapeutic agent.

Pharmaceutical compositions may be supplied as a kit comprising acontainer that comprises a fusion polypeptide or dimeric protein asdescribed herein. A therapeutic molecule can be provided, for example,in the form of an injectable solution for single or multiple doses, oras a sterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticprotein. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition.

The invention is further illustrated by the following non-limitingexamples.

Example 1

Molecule Design and Preparation: Two ApoA-1-Fc cDNA constructs weredesigned, synthesized, expressed by transient transfection of COSTcells, and the expressed proteins then purified by Protein Achromatography. One construct had the nucleotide sequence shown in SEQID NO:1 and encoded the fusion polypeptide of SEQ ID NO:2, and is alsoreferred to herein as ApoA-1(26)Fc or THER4. This construct contained aDNA segment encoding a 26 amino acid linker (residues 268-293 of SEQ IDNO:2) between the C-terminal end of human ApoA-1 (residues 1-267 of SEQID NO:2) and a human γ1 Fc variant (residues 294-525 of SEQ ID NO:2).Upon expression in mammalian cells and cleavage of the secretory signalpeptide (residues 1-18), and any potential cleavage of the propeptide(residues 19-24), this fusion polypeptide had a predicted amino acidsequence corresponding to residues 19-525, 19-524, 25-525, or 25-524 ofSEQ ID NO:2 (the C-terminal lysine of the Fc region is known to befrequently cleaved in the production of Fc-containing proteins). Theother construct contained ApoA-1 and Fc regions identical to those ofthe ApoA-1(26)Fc construct, but lacked a (gly4ser) linker between humanApoA-1 and the Fc regions; this construct is also referred to herein asApoA-1(2)Fc (Theripion) or as THER0 (for no (gly4ser) repeat units).This construct does contain a two amino acid linker due to insertion ofoverlapping restriction sites between the ApoA-1 region and the hingeregion of human IgG1.

Cholesterol efflux: The cholesterol efflux activity of the ApoA-I fusionproteins were measured using an in vitro assay. See Tang et al., J LipidRes. 47:107-14, 2006. In vitro cholesterol efflux assays were performedusing radio-labelled cholesterol and BHK cells expressing amifespristone-inducible human ABCA1. H3-cholesterol was added to growthmedia in order to label cellular cholesterol 24 hours prior totreatments, and ABCA1 is induced using 10 nM mifepristone for 16-20hours. Cholesterol efflux was measured by incubating cells with orwithout the fusion proteins for 2 hours at 37° C., chilled on ice, andmedium and cells separated to measure radiolabeled cholesterol.Wild-type human ApoA-1 protein was used as a positive control. Acommercially available ApoA-1-Fc protein, linked directly to Fc withoutany linker between the ApoA-1 and Fc regions (APOA1 Recombinant HumanProtein, hIgG1-Fc tag; Sino Biological, Inc.), was also tested and isreferred to herein as ApoA-1(0)Fc (Sino Biol). The results of this assayare shown in FIG. 1. Cholesterol efflux was increased in culturescontaining ApoA-1-Fc with a 26 amino acid linker (ApoA-1(26)Fc),compared to either ApoA-1-Fc with a two amino acid linker (ApoA-1(2)Fc(Theripion)) or ApoA-1-Fc without a linker (ApoA-1(0)Fc (Sino Biol)).ApoA-1(26)Fc also had activity similar to wild-type human ApoA-1(Control ApoA-1).

Example 2: Generation of Fusion Constructs and Sequence Verification

Additional ApoA1 fusion constructs were designed and the fusion genesequences were submitted to Blue Heron (Bothell, Wash.) for genesynthesis. A basic schematic diagramming the position of functionaldomains is shown in FIGS. 2A and 2B for the design of the ApoA1 fusionproteins. Fusion gene constructs inserted into pUC-based vectorsisolated by restriction enzyme digestion, and fragments encoding thefusion genes were subcloned into the mammalian expression vector pDG.Briefly, HindIII+XbaI flanking restriction sites were used for removalof each expression gene from the vector, subfragments isolated by gelelectrophoresis, DNA extracted using QIAquick purification columns, andeluted in 30 microliters EB buffer. Fragments were ligated intoHindIII+XbaI digested pDG vector, and ligation reactions transformedinto NEB 5-alpha, chemically competent bacteria. Clones were inoculatedinto 3 ml LB broth with 100 μg/ml ampicillin, grown at 37° C. overnightwith shaking at 200 rpm, and plasmid DNA prepared using the QIAGEN spinplasmid miniprep kits according to manufacturer's instructions.Sequencing primers were obtained from IDT Integrated DNA Technologies(Coralville, Iowa) and included the following:

pdgF-2: (SEQ ID NO: 16) 5′-ggttttggcagtacatcaatgg-3′; pdgR-2:(SEQ ID NO: 17) 5′-ctattgtcttcccaatcctccc-3′; higgras: (SEQ ID NO: 18)5′-accttgcacttgtactcctt-3′.

Plasmid DNA (800 ng) and sequencing primers (25 pmol, or 5 μl of a 5pmol/μl stock) were mixed and submitted for DNA sequencing by GENEWIZ(South Plainfield, N.J.). Chromatograms were then analyzed, sequencesassembled into contigs, and sequence verified using Vector Nti Advance11.5 software (Life Technologies, Grand Island, N.Y.).

Example 3: Expression of Fusion Proteins in a Transient HEK 293TTransfection System

This example illustrates transfection of plasmid constructs andexpression of fusion proteins described herein in a mammalian transienttransfection system. The Ig fusion gene fragments with correct sequencewere inserted into the mammalian expression vector pDG, and DNA frompositive clones was amplified using QIAGEN plasmid preparation kits(QIAGEN, Valencia, Calif.). Five different constructs were generated.These each included the native coding sequence of the human ApoA-1 gene(nucleotide sequence shown in SEQ ID NO:35, encoding the amino acidsequence shown in SEQ ID NO:36). Each sequence included the wild-typesignal peptide (nucleotides 1-54 of SEQ ID NO:35, encoding amino acids1-18 of SEQ ID NO:36) and propeptide sequences (nucleotides 55-72 of SEQID NO:35, encoding amino acids 19-24 of SEQ ID NO:36) for apolipoproteinA-1. The C-terminal Q (Gln) residues of the ApoA-1 sequence was linkedvia a variable length linker segment to the human IgG1 hinge, CH2, andCH3 domains to create a single chain (ApoA-1)-lnk-human IgG1 Fc fusiongene/protein. The hinge sequence of the human IgG1 is mutated so thatthe three cysteines are substituted with serine residues, eliminatingdisulfide bond formation in this region or unpaired cysteines that mightcompromise proper folding of the fusion protein. The P238 and P331residues of CH2 are also mutated to serines to eliminate effectorfunctions such as ADCC and complement fixation. Each construct alsoincluded a linker sequence inserted between the carboxyl terminus ofapolipoprotein A-1 (ending with the sequence . . . TKKLNTQ (SEQ ID NO:35residues 261-267)) and the beginning of the hinge sequence of the humanFc (starting with the motif . . . EPKSSDKT . . . (SEQ ID NO:2 residues294-301). This linker sequence ranged from two amino acids (or four ifthe overlap with the flanking domains is included) to 36 amino acids inlength, depending on the construct.

The shortest linker included only two overlapping restriction sites(BglII and XhoI) with a linker length of six additional nucleotides ortwo additional non-native amino acids. The restriction sites wereincorporated into the coding sequence of the molecule so that only twoadditional amino acids needed to be added to the amino acid sequence.The BglII site of the linker overlaps with the codon for the C-terminalglutamine of ApoA-1, and three of the nucleotides encoding the XhoI siteform the codon for the first amino acid of the hinge (E-glutamic acid).The linker amino acid sequence (including the two overlapping aminoacids) is shown in residues 267-270 of SEQ ID NO:20, which is encoded bynucleotides 816-825 of SEQ ID NO:19. The fusion gene and protein forthis construct are identified as THER0 (since there are no (gly4ser)repeat units present) or apoA-1-lnk(2)hIgG. The nucleotide and aminoacid sequences for THER0 are listed as SEQ ID NO:19 and SEQ ID NO:20.The figures use the THER0 nomenclature to specify this construct.

The second construct included a linker that encodes two (gly4ser)sequences flanked by restriction sites (16 amino acid linker), and thefusion gene and protein for this construct are identified as THER2 (orapoA-1-lnk(16)-hIgG1 or apoA-1-(g4s)2-hIgG1). The nucleotide and aminoacid sequences for THER2 are listed as SEQ ID NO:21 and SEQ ID NO:22,respectively. The (gly4ser)2 linker sequence is shown in residues268-283 of SEQ ID NO:22, and the encoding nucleotide sequence for the(gly4ser)2 linker is shown in residues 817-864 of SEQ ID NO:21.

The third construct included a linker that encodes four (gly4ser)sequences flanked by restriction sites (26 amino acid linker), and thefusion gene and protein for this construct is identified as THER4 (orapoA-1-(g4s)4-mthIgG or apoA-1-lnk(26)-mthIgG), where “4” in “THER4”refers to the number of (gly4ser) repeat units, and the number 26 refersto the total number of amino acids encoded in the non-native, introducedlinker sequence. The nucleotide and amino acid sequences for THER4 arelisted as SEQ ID NO:1 and SEQ ID NO:2, respectively. The (gly4ser)4linker sequence is shown in SEQ ID NO:50 (residues 268-293 of SEQ IDNO:2), and the encoding nucleotide sequence for the (gly4ser)4 linker isshown in SEQ ID NO:49 (residues 817-894 of SEQ ID NO:1).

The fourth construct included a linker that encodes six (gly4ser)sequences flanked by restriction sites (36 amino acid linker), and thefusion gene and protein for this construct is identified as THER6 (orapoA-1-(g4s)6-mthIgG or apoA-1-lnk(36)-mthIgG). The nucleotide and aminoacid sequences for THER6 are listed as SEQ ID NO:23 and SEQ ID NO:24,respectively. The (gly4ser)6 linker sequence is shown in SEQ ID NO:52(residues 268-303 of SEQ ID NO:24), and the encoding nucleotide sequencefor the (gly4ser)6 linker is shown in SEQ ID NO:51 (residues 817-924 ofSEQ ID NO:23).

The fifth construct included a linker that encodes four (gly4ser)sequences flanked by restriction sites (36 amino acid linker), but inaddition, the construct included a second linker and an enzyme sequenceat the carboxyl terminus of the IgG1 domain. The (gly4ser)4 linkersequence is as described above for THER4 (nucleotide and amino acidsequences shown in SEQ ID NO:49 and SEQ ID NO:50, respectively). Thesecond linker is an 18 amino acid long sequence (VDGASSPVNVSSPSVQDI;amino acid residues 1-18 of SEQ ID NO:8, encoded by nucleotides 1-54 ofSEQ ID NO:7) that includes an N-linked glycosylation site, followed by asequence that encodes human RNase1 enzyme activity. The linker sequenceis listed as the first 54 nucleotides of SEQ ID NO 7, or the first 18amino acids of SEQ ID NO 8, followed by the RNase sequence. TheApoA-1-lnk-hlgGl segment is fused to the NLG-RNase, and this constructis identified as THER4RNA2. The nucleotide and amino acid sequences areidentified as SEQ ID NO:3 and SEQ ID NO:4, respectively.

Miniprep DNA for each of the five constructs was prepared and theconcentration checked by Nanodrop analysis.

The day before transfection, approximately 1.2×10⁶ 293 T cells wereplated to 60 mm dishes. Mini-plasmid preparations (4.0 μg DNA for 60 mmplates) were used for 293T transfections using the QIAGEN POLYFECT®reagent (Catalog #301105/301107) and following the manufacturer'sinstructions. Culture supernatants were harvested 48-72 hours aftertransfection. For most transfections, media was changed to serum-freemedia 24 hours after transfection, and cultures incubated for a further48 hours prior to harvest.

Culture supernatants were used directly for further analysis. 7 μl ofeach serum-free culture supernatant from transiently transfected cellswas loaded onto gels with a 4× dilution of 4×LDS sample buffer (LifeTechnologies, Grand Island, N.Y.) added to each sample to give a finalconcentration of 1×LDS loading buffer. For reducing gels, samplereducing agent was added to 1/10 final volume. Samples were heated at72° C. for 10 minutes and loaded on NuPAGE® 4-12% Bis-Tris gels (LifeTechnologies/ThermoFisher Scientific, Grand Island, N.Y.). Gels weresubjected to electrophoresis in 1×NuPAGE® MOPS SDS-PAGE running buffer(NP0001, Life Technologies/ThermoFisher) at 180 volts for 1.5 hours, andproteins transferred to nitrocellulose using the XCell II™ Blot Module(Catalog #EI002/EI9051, Life Technologies/ThermoFisher, Grand Island,N.Y.) at 30 volts for 1 hour. Blots were blocked overnight at 4° C. inPBS containing 5% nonfat milk Blots were incubated with 1:250,000×dilution of horseradish peroxidase conjugated goat anti-human IgG(Jackson Immunoresearch, Catalog #109-036-098, Lot #122301). Blots werewashed three times for 30 minutes each in PBS/0.05% Tween 20, and weredeveloped in ThermoScientific ECL reagent (Catalog #32106) for 1 minute.Blots were exposed to autoradiograph film for 30 seconds to 2 minutes,depending on the blot. FIG. 3 shows Western Blot analysis of culturesupernatants from representative 293T transient transfections. Positiveand negative controls (CD40IgG and mock transfection/no DNA,respectively) were included in each transfection series. Transfectedsamples are as indicated in FIG. 3; lanes from left to right are asfollows: Lane #1—mock transfection; Lane #2—CD40IgG; Lane #3—MW markers;Lane #4—THER0; Lane #5—THER2; Lane #6—THER4; Lane #7—THER6; Lane #8—MWmarker; Lane #9—THER4RNA2.

The THER0, THER2, THER4, and THER6 fusion proteins run at a positionabove the 50 kDa molecular weight marker. The predicted molecular weightfor these fusion proteins should be approximately 55, 56, 56.6, and 57kDa, respectively. The increasing linker length is evident by alteredmobility for each fusion protein. The THER4RNA2 molecule is predicted tobe approximately 73.2 kDa, while ApoA-1 is predicted to run at 28.6 kDa.The CD40IgG control is expected to run at approximately 55 kDa.

Example 4: Expression of THERmthIgG and Multi-subunitIg FusionConstructs and Fusion Proteins in Stable CHO Cell Lines

This example illustrates expression of the different Ig fusion genesdescribed herein in eukaryotic cell lines and characterization of theexpressed fusion proteins by SDS-PAGE and by IgG sandwich ELISA.

Transfection and Selection of Stable Cell Lines Expressing FusionProteins

Stable production of the Ig fusion protein was achieved byelectroporation of a selectable, amplifiable plasmid, pDG, containingthe THER-mthlgG cDNAs (human apo A-1 forms separated from the hinge andFc domain of human IgG1 by linkers of varying lengths) under the controlof the CMV promoter, into Chinese Hamster Ovary (CHO) CHO DG44 cells.

The pDG vector is a modified version of pcDNA3 encoding the DHFRselectable marker with an attenuated promoter to increase selectionpressure for the plasmid. Plasmid DNA (200 μg) was prepared using QIAGENHISPEED® maxiprep kits, and purified plasmid was linearized at a uniqueAscl site (New England Biolabs, Ipswich, Mass. Catalog #R0558), purifiedby phenol extraction (Sigma-Aldrich, St. Louis, Mo.), ethanolprecipitated, washed, and resuspended in EX-CELLO 302 tissue culturemedia, (Catalog #14324, SAFC/Sigma Aldrich, St. Louis, Mo.). Salmonsperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as carrier DNA justprior to phenol extraction and ethanol precipitation. Plasmid andcarrier DNA were coprecipitated, and the 400 μg was used to transfect2×10⁷ CHO DG44 cells by electroporation.

For transfection, CHO DG44 cells were grown to logarithmic phase inEX-CELLO 302 media (Catalog #13424C, SAFC Biosciences, St. Louis, Mo.)containing glutamine (4 mM), pyruvate, recombinant insulin (1 μg/ml),penicillin-streptomycin, and 2×DMEM nonessential amino acids (all fromLife Technologies, Grand Island, N.Y.), hereafter referred to as“EX-CELL 302 complete” media. Media for untransfected cells and cells tobe transfected also contained HT (diluted from a 100× solution ofhypoxanthine and thymidine) (Invitrogen/Life Technologies, Grand Island,N.Y.). Electroporations were performed at 280 volts, 950 microFarads,using a BioRad (Hercules, Calif.) GENEPULSER® electroporation unit withcapacitance extender. Electroporation was performed in 0.4 cm gapsterile, disposable cuvettes. Electroporated cells were incubated for 5minutes after electroporation prior to transfer of culture tonon-selective EX-CELL 302 complete media in T75 flasks.

Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 250 cells/well (2500 cells/ml)to 2000 cells/well (20,000 cells/ml). Culture media for cell cloning wasEX-CELL 302 complete media containing 50 nM methotrexate. Transfectionplates were fed at five day intervals with 80 μl fresh media. After thefirst couple of feedings, media was removed and replaced with freshmedia. Plates were monitored and individual wells with clones were feduntil clonal outgrowth became close to confluent, after which cloneswere expanded into 24 well dishes containing 1 ml media. Aliquots of theculture supernatants from the original 96 well plate were harvested to asecond 96 well plate prior to transfer and expansion of the cells in 24well plates. This second plate was frozen until ELISA analysis toestimate IgG concentrations.

Screening Culture Supernatants for Production Levels of RecombinantFusion Proteins

Once clonal outgrowth of initial transfectants was sufficient, serialdilutions of culture supernatants from master wells were thawed and thedilutions screened for expression of Ig fusion protein by use of an IgGsandwich ELISA. Briefly, NUNC MAXISORP® plates were coated overnight at4° C. with 2 μg/ml F(ab′2) goat anti-human IgG (Jackson Immunoresearch,West Grove, Pa.; Catalog #109-006-098) in PBS. Plates were blocked inPBS/3% BSA, and serial dilutions of culture supernatants incubated atroom temperature for 2-3 hours or overnight at 4° C. Plates were washedthree times in PBS/0.05% Tween 20, and incubated with horseradishperoxidase conjugated F(ab′2)goat anti-human IgG (JacksonImmunoresearch, West Grove, Pa., Catalog #109-036-098) at1:7500-1:10,000 in PBS/0.5% BSA, for 1-2 hours at room temperature.Plates were washed five times in PBS/0.05% Tween 20, and bindingdetected with SUREBLUE RESERVE™ TMB substrate (KPL Labs, Gaithersburg,Md.; catalog #53-00-02). Reactions were stopped by addition of equalvolume of 1N HCl, and absorbance per well on each plate was read at 450nm on a SYNERGY™ HT plate reader (Biotek, Winooski, Vt.). Concentrationswere estimated by comparing the OD450 of the dilutions of culturesupernatants to a standard curve generated using serial dilutions of aknown standard, a protein A purified human IgG fusion protein with an Igtail identical to that of the THER clones. Data was collected andanalyzed using GENS™ software (Biotek, Winooski, Vt.) and MicrosoftOffice EXCEL® spreadsheet software.

The results of initial screening of the CHO transfectants are summarizedin Table 6 and FIGS. 4A-4E and 5A-5C. Table 6 shows a summary of thenumber of clones screened, the range of expression levels observed frominitial 96-well cultures, and the expression observed from initial T25and/or 24 well spent cultures. FIGS. 4A-4E show a series of columnargraphs representing the production levels obtained from each CHO cloneof a transfection series. The clones from the THER0, THER2, THER4,THER6, and THER4RNA2 transfections are displayed as a group in each ofthe five panels shown. Each clone was screened at least once by IgGsandwich ELISA to assess expression level of the fusion protein. FIGS.5A-5C show three panels showing the results of 6 and 10 day assays offusion protein expression from the CHO transfectants with the highestexpression after initial screening. Six and ten day assays wereperformed by setting up 5 ml cultures at 1×10⁵ viable cells/ml (5×10⁵initial inoculum) in T25 flasks. The cultures were grown for six daysafter which a 1 ml aliquot was removed and live and dead cells counted.Cells were then centrifuged and the culture supernatants saved forfurther analysis by IgG sandwich ELISA and other analyses. The remainderof the cultures were incubated for a further four days until day 10, andthe cells counted for cell number, viability, and a supernatant sampleharvested for IgG sandwich ELISA. The results are tabulated in columnarform for each clone as shown in the graphs for cell number at day 6 andday 10, viability, and concentration of fusion protein.

TABLE 6 Expression of ApoA1-IgG fusion proteins in stably transfectedCHO DG44 cells 6/10 day 96 well sups Spent T25 assay on Clonesexpression top producer top clones Construct Screened range (μg/ml)(μg/ml) (μg/ml) THER0 45   0-46.5 135 60/230 THER2 135 0-36 145 45/125THER4 237 0-76 165 70/200 THER6 192 0-57 145 85/250 THER4RNA2 50 0-45 9050/118

The clones with the highest production of the fusion protein wereexpanded into T25 and then T75 flasks to provide adequate numbers ofcells for freezing and for scaling up production of the fusion protein.Production levels were further increased in cultures from the four bestclones by progressive amplification in methotrexate-containing culturemedia. At each successive passage of cells, the EX-CELL 302 completemedia contained an increased concentration of methotrexate, such thatonly the cells that amplified the DHFR plasmid could survive. Media fortransfections under selective amplification contained varying levels ofmethotrexate (Sigma-Aldrich) as selective agent, ranging from 50 nM to 1μM, depending on the degree of amplification achieved.

Purification of Fusion Proteins from Culture Supernatants

Supernatants were collected from spent CHO cell cultures expressing theApo A-1-lnk-mthIgG1 construct, filtered through 0.2 μm PES expressfilters (Nalgene, Rochester, N.Y.) and subjected to gravity flowaffinity chromatography over a Protein A-agarose (IPA 300 crosslinkedagarose, or IPA 400HC crosslinked agarose) column (Repligen, Waltham,Mass.). The column was conditioned with 0.1M citrate buffer, pH2.2, thensupernatant adjusted to pH 8.0 with 0.5M NHCO3, and loaded by gravityflow to allow binding of the fusion proteins. Columns were then washedwith several column volumes column wash buffer (90 mM Tris-Base, 150 mMNaCl, 0.05% sodium azide, pH 8.7) or Dulbecco's modified PBS, pH 7.4prior to elution. Bound protein was eluted using 0.1 M citrate buffer,pH 3.2. Fractions (0.8-0.9 ml) were collected into 0.2 ml 0.5MNaCO₃—NaHCO₃ buffer to neutralize each fraction. Protein concentrationof aliquots (2 μl) from each fraction were determined at 280 nM using aNanodrop (Wilmington Del.) microsample spectrophotometer, with blankdetermination using 0.1 M citrate buffer, pH 3.2, 0.5M NaCO₃ at a 10:1v:v ratio. Fractions containing fusion protein were pooled, and bufferexchange performed by dialysis using Spectrum Laboratories G2 (RanchDominguez, Calif., Catalog #G235057, Fisher Scientific catalog#08-607-007) FLOAT-A-LYZER® units (MWCO 20 kDa) against D-PBS (Hyclone,ThermoFisher Scientific, Dallas, Tex.), pH 7.4. Dialysis was performedin sterile, 2.2 liter Corning roller bottles at 4° C. overnight.

After dialysis, protein was filtered using 0.2 μM filter units, andaliquots tested for endotoxin contamination using PYROTELL® LAL gel clotsystem single test vials (STV) (Catalog # G2006, Associates of Cape Cod,East Falmouth, Mass.). The predicted OD 280 of a 1 mg/ml solution of theTHER4 fusion protein was determined to be 1.19 (mature protein withouteither the signal peptide or the 6 amino acid propeptide) or 1.27(including the 6 amino acid propeptide) using the protein analysis toolsin the VECTOR NTI® Version 11.5 Software package (Informax, NorthBethesda, Md.) and the predicted cleavage site from the online ExPASyprotein analysis tools. It is unclear whether the fusion proteinsecreted from the CHO cells is homogeneous with regard to completecleavage of the propeptide from the recombinant molecules. The OD280 foreach purified fusion protein was corrected using these tools.

Reducing and Nonreducing SDS-PAGE Analysis of apo A-1 Ig Fusion Proteins

Purified fusion proteins were analyzed by electrophoresis onSDS-Polyacrylamide 4-12% Bis-Tris NuPAGE® gels (Life Technologies, GrandIsland, N.Y.). Fusion protein samples were heated at 72° C. for 10minutes in LDS sample buffer with and without reduction of disulfidebonds and applied to 4-12% BIS-Tris gels (Catalog #NP0301, LIFETechnologies, Grand Island, N.Y.). Five micrograms of each purifiedprotein was loaded on the gels. The proteins were visualized afterelectrophoresis by IMPERIAL™ protein staining (Pierce Imperial ProteinStain Reagent, Catalog #24615, ThermoFisher Scientific/Pierce, Rockford,Ill.), and destaining in distilled water. Molecular weight markers wereincluded on the same gel (KALEIDOSCOPE™ Prestained Standards, Catalog#161-0324, Bio-Rad, Hercules, Calif.). The results from representativenonreducing and reducing gels are shown in FIGS. 6A and 6B,respectively. Lanes are as follows from left to right: Lane#1—KALEIDOSCOPE prestained MW markers; Lane #2—THER0; Lane #3—THER2;Lane #4—THER4; Lane #5—THER6; Lane #6—THER4RNA2; Lane #7—KALEIDOSCOPEPrestained MW markers. Approximate molecular weights are indicated onthe figures.

Again, the linker length difference between the different fusionproteins is evident on both the reducing and nonreducing gels, with theTHER0 protein running at just over 50 kDa. The absence of hingedisulfides is evident by the similar mobility for each protein whenelectrophoresed under reducing or nonreducing conditions.

Native Gel Electrophoresis of apo A-1 IgG Fusion Proteins

The protein A purified fusion proteins were subjected to native PAGEanalysis. BLUE Native PAGE gels were run using 4-16% Bis-TrisNativePAGE™ gels (Life Technologies/ThermoFisher) with cathode and anodebuffers prepared according to manufacturer's instructions. Samples (4.5μg each fusion protein) were prepared without heating, using 4× samplebuffer, without detergents. Gels were run for 30 minutes at 150 volts,followed by 1 hour at 180 volts, and the final hour at 220 volts. Gelswere washed in distilled water and incubated for two hours in IMPERIAL™Protein stain. Gels were extensively destained overnight with repeatedwashes in distilled water to remove the blue dye present in the cathodebuffer used for running the gels. FIG. 7 shows a representative nativegel using these conditions. Molecular weight markers were GE Healthcarehigh molecular weight calibration markers, a mixture of six large,multicomponent proteins, resuspended in the loading buffer used forsamples, again without added detergents. Samples were loaded as follows:Lane #1—ORENCIA® (abatacept; CTLA4hIgG); Lane #2—anti-mouse CD40monoclonal antibody 1C10; Lane #3—THER4RNA2; Lane #4—GE Healthcare HighMW calibration markers; Lane #5—THER6; Lane #6—THER4; Lane #7—THER2;Lane #8—THER0; Lane #9—GE Healthcare high MW markers; Lane #10—AthensResearch Apo A-1.

The native ApoA1-IgG fusion proteins run at an approximate molecularweight somewhere between the 140 and 233 kDa markers and with a similarmobility as ORENCIA® (abatacept), a CTLA4Ig fusion protein with the samehuman IgG1 Fc domain. The THER4RNase bispecific fusion protein did notstain well with the IMPERIAL stain possibly due to the highly basiccomposition of the RNase domain, but appears to migrate in a morediffuse pattern with the predominate visible band migrating between the233 and 440 kDa standards.

Example 5: Use of an IgG/Apo A-1 Sandwich ELISA to Assess Binding ofTHER Apo A-1 Fusion Proteins

An antigen binding ELISA was performed to assess the ability of Igfusion proteins, captured by immobilized anti-human IgG (Fc-specific) tobind to and be detected by a horseradish peroxidase conjugated antibodyspecific for human apolipoprotein A-1. High protein-binding, 96-wellELISA plates (NUNC MAXISORP® plates, ThermoFisher Scientific) werecoated with 1.5 kg/ml goat anti-human IgG (Jackson Immunoresearch).Plates were blocked overnight at 4° C. with PBS/3% BSA. Serial dilutionsof each THER fusion protein, starting at 5 μg/ml, were incubatedovernight at 4° C. The plate was washed three times and then incubatedwith horseradish peroxidase conjugated anti-human apolipoprotein A-1(ThermoFisher Scientific, catalog #PAI-28965) diluted 1:1500. Plateswere incubated at room temperature for 2 hours. Plates were washed fourtimes, then SUREBLUE RESERVE™ TMB substrate (Catalog #: 53-00-02, KPL,Gaithersburg, Md.) was added to the plate at 80 μl/well. Development wasstopped by addition of 80 μl/well 1N HCl. Samples were read at 450 nmusing a SYNERGY™ HT Biotek plate reader (Biotek Instruments, Winooski,Vt.) and data analyzed using GENS™ 2.0 software.

FIG. 8 shows the results from a representative Apo A-1 binding ELISA.OD450 is plotted versus concentration of fusion protein. THER 0, 2, 4,6, and THER4RNA2 fusion proteins all exhibited similar dose-responsecurves, indicating that the molecules can each be captured by binding tothe Ig tail and detected by binding of the Apo A-1 domain to theantibody targeted to human Apo A-1. Human apolipoprotein A-1 (AthensResearch & Technology, catalog #16-16-120101) was included as a controland was not captured by the anti-human Fc specific antibody. At higherconcentrations, the molecule showed weak binding by the antibodytargeted to Apo A-1, indicating that the Apo A-1 may have bound weaklyto the plastic without capture by the anti-Fc antibody.

Example 6: Expression and Testing of an RNase Bifunctional Enzyme LipidTransport Fusion Molecule

For the Apo A-1 IgG RNase fusion protein (THER4RNA2), RNase activity wasassayed to determine whether fusion of the enzyme to the carboxyl end ofthe fusion construct interfered with ability of the molecule to digestRNA. FIGS. 9 and 10 show the results of an RNASEALERT™ assay (IDT,Coralville, Iowa) performed using the fluorescence and kinetic assayfunctions of the SYNERGY™ HT plate reader. RNASEALERT™ Substrate is asynthetic RNA oligonucleotide that has a fluorescein (R) on one end anda dark quencher (Q) on the other end. When intact, the substrate haslittle or no fluorescence, but when cleaved by an RNase, the substratefluoresces green (490 nm excitation, 520 nm emission) and can bedetected with the appropriately equipped fluorescence plate reader. Apositive signal in this assay shows increasing fluorescence signal overtime due to cleavage of the substrate by RNase present in the sample(s).Microplates were incubated with RNASEALERT substrate (a fixedconcentration of 20 pmol/μl), 1×RNASEALERT buffer, and fusion protein orenzyme controls dilutions added to each well of a 96 well plate. Enzymeactivity assays were performed in triplicate for each sample, and thekinetic assay allowed to proceed for 45 minutes, with successivereadings every 60 seconds. The increasing fluorescence at each timepoint is displayed for each well as a trace of RFU/well as a function oftime in FIG. 9. Serial dilutions of enzyme/fusion protein included 20pmol/μl, 13.4 pmol/μl, 8.9 pmol/μl, 6 pmol/μl, 4 pmol/μl, 2.7 pmol/μl,1.8 pmol/μl, and no enzyme. RNase A (Ambion/ThermoFisher, catalog#AM2270) was included as a positive control, and THER4 (apoA-1-lnk26-hIgG) was included as a negative control for comparing to theTHER4RNA2 fusion protein. Overlays of the traces generated using the 4pmol/μl enzyme are shown in FIG. 10. Two replicates of the RNaseA,THER4RNA2, and THER4, are shown. All enzymes are at 4 pmol/μl and thesubstrate is present at 20 pmol/μl.

Example 7: Measurement of Cholesterol Efflux to Fusion Protein Acceptors

Using two separate assays, THER0, THER2, THER4, THER6, and THER4RNA2fusion proteins were assessed for their ability to act as acceptormolecules for reverse cholesterol transport from pre-loadedmonocyte/macrophage mammalian cell lines. The first assay used the humanmonocytic/macrophage cell line THP-1 and a fluorescently labeledderivative of cholesterol, BODIPY-cholesterol or TOPFLUOR-cholesterol(cholesterol compound with fluorescent boron dipyrromethene difluoridelinked to sterol carbon-24) (Avanti Polar Lipids, Alabaster, Ala.). TheTHP-1 cells were grown in RPMI with 4 mM glutamine, 10% FBS andmaintained in mid-logarithmic growth prior to plating. The protocol wasadapted from the procedures outlined in Sankaranararyanan et al. (J.Lipid Res. 52:2332-2340, 2011) and Zhang et al. (ASSAY and DrugDevelopment Technologies: 136-146, 2011). Cells were harvested andplated to 96-well flat bottom tissue culture plates at 2×10⁶ cells/ml or2×10⁵ cells/well in 100 μl RPMI media containing 33 ng/ml PMA. Cellswere maintained in culture for 36-48 hours to allow for differentiationto occur prior to the assay. Culture media was aspirated and plates werewashed in 1×PBS. Labeling media consisting of the following components(Phenol Red free RPMI with media supplements, 0.2% FBS, with ACATinhibitor at 2 μg/ml, Sandoz 58-035 (Sigma-Aldrich, St. Louis Mo.), LXRagonist TO-901317 at 2.5 μM (Sigma-Aldrich, St. Louis, Mo.), 35 ng/mlPMA (Sigma-Aldrich, St. Louis, Mo.), and 1.25 mM methylbeta-cyclodextrin (Sigma-Aldrich, St. Louis, Mo.), 50 uM cholesterol(Sigma-Aldrich, St. Louis, Mo., and 25 μM TOPFLUOR cholesterol (AvantiPolar Lipids, Alabaster, Ala.) was added at a volume of 100 μl/well andincubated at 37 C, 5% CO2 for 10-12 hours. Equilibration media, RPMIcomplete with 10% FBS, 33 ng/ml PMA (100 ul/well), was added to eachwell and incubated for 8 hours prior to incubation with acceptors.Labeling/equilibration media was aspirated from plates, and plates werewashed twice with 200 μl/well PBS+0.15% BSA. Efflux acceptor reagentswere added to individual wells in efflux buffer and incubated for twohours prior to assay. Acceptors were added to efflux buffer atconcentrations ranging from 100 nM to 500 nM, depending on the assay.Efflux buffer was phenol red-free RPMI with growth supplements and 0.15%BSA. Samples were run in sets of 6-12 per condition/acceptor, and aminimum of five replicates used for statistical analysis. APO A-1 wasrun as a positive control, and efflux media alone was used as thebackground negative control (baseline efflux). The efflux reaction wasallowed to proceed for two hours, after which culture media washarvested to black, flat bottom 96-well plates (media reading). Celllysates were prepared by addition of 100 ul 0.1 N NaOH to each well ofthe efflux plate, and incubation for 15 minutes on a plate shaker at 4°C. Cell lysates were transferred to black, 96-well plates (lysatereading), and fluorescence for media and lysate samples measured using aSYNERGY™ HT plate reader with excitation at 485 nm and emission at 528nm. Efflux was calculated as the ratio of the fluorescence measurements:(media/(media+lysate)×100). The specific efflux was calculated bysubtracting the baseline readings of the samples with no acceptorpresent from the total efflux/sample for each tested acceptor. Dataanalysis was performed using GraphPad Prism v 4.0 Software (San Diego,Calif.). The assay results are shown in FIG. 11.

The second assay used the mouse macrophage cell line J774A.1 (ATCC,Manassas, Va.) to assess reverse cholesterol transport (RCT) using aradioactive derivative of cholesterol, [³H]-cholesterol as described bySankaranararyanan et al. (J. Lipid Res. 52:2332-2340, 2011) and Yanceyet al. (J. Lipid Res. 45:337-346, 2004). Briefly, J774 cells (3.5×10⁵per well in 24 well plates) were incubated for 24 hours in 0.25 ml RPMImedia supplemented with 5% FBS, ACAT inhibitor Sandoz 58-035 (2 μg/ml),and 4 μUCi/ml of [³H]-cholesterol. ACAT inhibitor was present at alltimes during the assay. Cells were equilibrated 16-24 hours in mediawith or without cAMP (0.3 mM) prior to incubation with acceptors. Thepresence of cAMP upregulates the ABCA1 molecule. Labeled cells werewashed in media containing 1% BSA, then acceptor molecules were added at50, 100 and 200 nM in MEM-HEPES media and incubated for 4 hours prior tomeasurements. All treatments were performed in triplicate. The [³H]cholesterol in 100 μl of the media was then measured by liquidscintillation counting. The percentage efflux is based on the total[³H]cholesterol present in the cells before the efflux incubation (tosample). To measure the [³H]cholesterol present in the cells, the celllipids were extracted by incubating the cell monolayers overnight inisopropanol. After lipid extraction, the total [³H]cholesterol presentin the lipid extract was measured by liquid scintillation counting. Dataanalysis was performed using GraphPad Prism software 4.0 (San Diego,Calif.). The assay results are shown in FIG. 12.

Example 8: Construction of a PON1 Bifunctional Enzyme Lipid TransportFusion Molecule

In addition to the apoA-1-IgG-RNase expression constructs describedabove, additional molecules which physically link the ApoA-1phospholipid transport function to the active sites of other enzymedomains are constructed. One such molecule contains a segmentcorresponding to human paraoxanase 1 (PON1), with nucleotide and encodedamino acid sequences as shown in SEQ ID NO:11 and SEQ ID NO:12,respectively. This arylesterase enzyme is present in human serumexclusively associated with high density lipoprotein (HDL), and inhibitsoxidation of low density lipoprotein molecules. This protection fromoxidation also inhibits development of vascular and coronary diseases.The mature protein form of PON1 is unique in that it retains its aminoterminal signal peptide after secretion (amino acid residues 1 to 15 ofSEQ ID NO:12, encoded by nucleotide residues 1 to 45 of SEQ ID NO:11).Expression of a mutant form of PON1 with a cleavable amino terminusdemonstrated that PON1 associates with lipoproteins through its aminoterminus by binding to phospholipids directly rather than first bindingto ApoA-1. See Sorenson et al., Arteriosclerosis, Thrombosis, andVascular Biology 19:2214-2225, 1999. Removal of the signal sequence wasfound to eliminate binding of the PON1 moiety to phospholipids,proteoliposomes, and serum lipoproteins. Additionally, in the absence ofphospholipid, wild-type PON1 does not bind directly to ApoA-1. SeeSorenson et al., supra. These PON1 signal sequence mutants showedreduced enzyme activity, possibly due to inability to bind the optimalphospholipid substrates. Nevertheless, a recombinant, active form ofhuman PON1 has been expressed in bacteria that is missing this signalsequence. See Stevens et al., Proc. Natl. Acad. Sci. USA105:12780-12784, 2008. The presence of ApoA-1 does appear to stabilizearylesterase activity of the enzyme.

Removal of the amino terminal signal sequence of PON1 (and thereby thephospholipid binding moiety) and substitution of this region with humanapoA-1-lnk-IgG directly links the enzyme activity with the phospholipidbinding domain of ApoA-1, stabilizing the arylesterase enzyme activitywhile providing the optimal substrates bound to the ApoA-1 domain. Sucha molecule still traffics and is transported with the phospholipidsbound by ApoA-1 and retains enzyme activity due to replacement of thesignal sequence domain with an alternative phospholipid binding domain.In addition, a bifunctional molecule fusing these two domains exhibitsimproved expression and facilitates targeting of the PON1 activity tothe choroid plexus through active binding of apo A-1. PON1 has beenexpressed at the carboxyl terminus of an insulin receptor targetedantibody (see Boado et al., Mol. Pharm. 5:1037-1043, 2008; Boado et al.,Biotechnology and Bioengineering 108:186-196, 2011); however, the aminoterminal signal peptide was included in this fusion protein. The fusiongene and protein described here provides a novel method of PON1 fusionprotein expression, eliminating the requirement for the signal peptideby a direct physical coupling of the truncated enzyme to the apo A-1domain, thereby preserving and stabilizing both the binding function andarylesterase activity of PON1.

Sequences for the fusion gene and protein are shown in SEQ ID NO:27 andSEQ ID NO:28 for THER4PON1 (nucleotide and amino acid sequences,respectively) and in SEQ ID NO:37 and SEQ ID NO:38 for THER2PON1(nucleotide and amino acid sequences, respectively). Similar fusiongenes and proteins contain alternative linker forms of ApoA-1 fused tothe hIgG1-linker-PON1 segment(s). The PON1 sequences within theTHER4PON1 and THER2PON1 molecules correspond to the Q192 allele forhuman PON1.

Alternative forms of PON1 are also used to construct bifunctional fusionmolecules linking PON1 to Apo A-1. A sequence polymorphism that affectsenzyme activity for different substrates is present at position 192 ofthe PON1 sequence. See Steven et al., supra. The amino acid at thisposition may be glutamine (Q) or arginine (R) in humans, or a lysine (K)in rabbits. The arginine allele at position 192 has been reported tohave a higher catalytic activity in vitro and in vivo. Similarly, therabbit form of PON1 with a lysine at position 192 has been reported tohave a more stable catalytic activity in vitro and in vivo (see Stevenet al., supra; Richter et al., Circulation Cardiovascular Genetics1:147-152, 2008). These alternative PON1 sequences are shown in SEQ IDNO:41 (nucleotide) and SEQ ID NO:42 (amino acid) for the PON1 Q192Kform, and SEQ ID NO:43 (nucleotide) and SEQ ID NO:44 (amino acid) forthe PON1 Q192R form. The fusion construct between these alternate PON1forms and the THER4 sequence (apoA-1(g4s)4hIgGNLG- . . . ) is designatedas THER4PON1 Q192K (nucleotide and amino acid sequences shown in SEQ IDNO:45 and SEQ ID NO:46, respectively) or THER4PON1 Q192R (nucleotide andamino acid sequences shown in SEQ ID NO:47 and SEQ ID NO:48,respectively), depending on the polymorphism present in the PON1sequence at amino acid 192 of the PON1 sequence (or at amino acid 720 ofthe THER4PON1Q variants shown in SEQ ID NO:46 and SEQ ID NO:48).Similarly, a THER2 form of the fusion gene/protein is indicated asTHER2PON1 Q192K or THER2PON1Q192R. For all of these fusion constructs,the PON1 amino terminal signal sequence (amino acids 1-15 of SEQ IDNO:12) is removed.

Bispecific Enzyme Lipoprotein Transfer Proteins comprising PON1 arescreened for arylesterase/PON1 activity using the nontoxic substrates4-(chloromethyl)phenyl acetate (CMPA) and phenyl acetate (see Richter etal., supra). These substrates are preferable for screening activitysince the substrate and reaction product are relatively nontoxiccompared to organophosphate pesticides. The CMPA substrate(Sigma-Aldrich, Inc. St Louis, Mo.) is incubated with serial dilutionsof fusion protein and rates of CMPA hydrolysis assayed at 280 nm for 4minutes at 25° C. using ultraviolet transparent 96-well plates (Costar,Cambridge, Mass.). Dilutions are run in triplicate or quadruplicate andsubstrate concentration fixed at 3 mmol/L in 20 mM Tris-HCl (pH 8.0),1.0 mM CaCl₂. Similarly, arylesterase assays are performed on phenylacetate as substrate. The rates of PA hydrolysis are measured at 270 nm,for 4 minutes under both high and low salt conditions.

Example 9: Construction of a PAFAH or CETP Bifunctional Enzyme LipidTransport Fusion Molecule

In addition to the apoA-1-IgG-RNase and apoA-1-IgG-PON1 expressionconstructs described above, additional molecules which physically linkthe ApoA-1 phospholipid transport function to the active sites of otherenzyme domains are constructed.

One such molecule contains a segment corresponding to human PAFAH(lipoprotein-associated phospholipase A2, human phospholipase A2 groupVII, platelet activating factor acetyl hydrolase), with nucleotide andencoded amino acid sequences as shown in SEQ ID NO:31 and SEQ ID NO:32,respectively (see also GenBank accession number NM_005084 (transcriptvariant 1)). The PAFAH amino acid sequence is encoded by nucleotides 270to 1592 of SEQ ID NO:31, with the STOP codon at nucleotides 1593 to1595. The fusion gene and protein are designed fusing the PAFAH codingsequence at the carboxyl end of the human IgG with the N-linkedglycosylation linker inserted between the two molecules. The THER4PAFAHnucleotide and encoded amino acid sequences are shown as SEQ ID NO:33and SEQ ID NO:34, respectively. The PAFAH sequence without the 21 aminoacid signal peptide (MVPPKLHVLFCLCGCLAVVYP; residues 1-21 of SEQ IDNO:32) is fused to the NLG linker at amino acid position 544 in SEQ IDNO 34.

Another such molecule contains a segment corresponding to human CETP orcholesteryl ester transfer protein (CETP), transcript variant 1, withnucleotide and encoded amino acid sequences as shown in SEQ ID NO:29 andSEQ ID NO:30, respectively (see also GenBank accession numberNM_000078). The CETP protein is encoded by nucleotides 58 to 1537 of SEQID NO:29. The fusion gene and protein are designed fusing the CETPcoding sequence at the carboxyl end of the human IgG with the N-linkedglycosylation linker inserted between the two molecules. The THER4CETP(or human apo A-1-(g4s)4-hIgG-NLG-CETP) nucleotide and encoded aminoacid sequences are shown as SEQ ID NO:39 and SEQ ID NO:40, respectively.The nucleotides (57-107 of SEQ ID NO 29) encoding the signal peptide(amino acids 1-17 of SEQ ID NO 30) are removed in order to create thefusion gene between the NLG linker sequence and the CETP mature peptide.The fusion junction between these two protein domains is located atamino acid 544 of SEQ ID NO 40.

Example 10: Construction and Expression of PON1 Bifunctional EnzymeLipid Transport Fusion Molecules

THER4PON1 (also referred to herein as “T4P1”) and otherApoA1-lnk-IgG-PON1 fusion gene variants were constructed by usingTHER4RNA2 as a recipient for substitution mutations, removing the RNasecontaining cassette and replacing it with different variants of the PON1sequence. PON1 cDNA constructs were designed in cassette form,synthesized by PCR amplification, and the sequences assembled bysubcloning using engineered restriction sites incorporated into themolecule design. Once cassettes were assembled in the desiredconfiguration, fusion gene constructs were assembled in pUC-basedvectors and final restriction fragments encoding the fusion genes weresubcloned into the mammalian expression vector pDG. Briefly,HindIII+XbaI or EcoRV flanking restriction sites were used for removalof each expression gene from the vector, subfragments isolated by gelelectrophoresis, DNA extracted using QIAquick purification columns, andeluted in 30 microliters EB buffer. Fragments were ligated intoHindIII+XbaI or EcoRV digested pDG vector, and ligation reactionstransformed into NEB 5-alpha, chemically competent bacteria. Clones wereinoculated into 3 ml LB broth with 100 μg/ml ampicillin, grown at 37° C.overnight with shaking at 200 rpm, and plasmid DNA was prepared usingthe QIAGEN spin plasmid miniprep kits according to manufacturer'sinstructions. Sequencing primers were obtained from IDT Integrated DNATechnologies (Coralville, Iowa) and included the following:

pdgF-2: (SEQ ID NO: 16) 5′-ggttttggcagtacatcaatgg-3′; pdgR-2:(SEQ ID NO: 17) 5′-ctattgtcttcccaatcctccc-3′; higgras: (SEQ ID NO: 18)5′-accttgcacttgtactcctt-3′.

For sequencing constructs, plasmid DNA (800 ng) and sequencing primers(25 pmol, or 5 μl of a 5 pmol/μl stock) were mixed and submitted for DNAsequencing by GENEWIZ (South Plainfield, N.J.). Chromatograms were thenanalyzed, sequences assembled into contigs, and sequence verified usingVector Nti Advance 11.5 software (Life Technologies, Grand Island,N.Y.). Clones with the correct sequence were then amplified and plasmidDNA was used for transient transfection of HEK293T cells using Polyfectreagent (QIAGEN, Valencia, Calif.), according to manufacturer'sinstructions. Transfection media was changed after 24 hours to a lowfluorescence, phenol red free media, FLUOROBRITE™ DMEM (LifeTechnologies, Grand Island, N.Y.), containing growth supplements(glutamine, pyruvate, non-essential amino acids, and pen/strep), but noserum. Serum-free transfection supernatants were harvested after 48-72hours. Proteins were analyzed by SDS PAGE, Western blotting, and ELISAanalysis. In addition, for proteins expressed at sufficient levels asdetermined by ELISA, undiluted and serial dilutions of culturesupernatants were filter-sterilized and used in further functionalassays.

Culture supernatants from transient and stable transfections were useddirectly for further analysis; in general, 10 μl of each serum-freeculture supernatant from transfected cells was loaded onto gels in 1×LDSsample buffer (Life Technologies, Grand Island, N.Y.). For reducinggels, sample reducing agent was added to 1/10 final volume. Samples wereheated at 72° C. for 10 minutes and loaded on NuPAGE® 4-12% Bis-Trisgels (Life Technologies/ThermoFisher Scientific, Grand Island, N.Y.).Gels were subjected to electrophoresis in 1×NuPAGE® MOPS SDS-PAGErunning buffer (NP0001, Life Technologies/ThermoFisher) at 180 volts for1.5 hours, and proteins transferred to nitrocellulose using the XCellII™ Blot Module (Catalog #E1002/EI9051, Life Technologies/ThermoFisher,Grand Island, N.Y.) at 30 volts for 1 hour. Blots were blocked overnightat 4° C. in PBS containing 5% nonfat milk Blots were incubated with1:100,000× dilution of horseradish peroxidase conjugated goat anti-humanIgG (Jackson Immunoresearch, Catalog #109-036-098, Lot #122301). Blotswere washed three times for 30 minutes each in PBS/0.05% Tween 20 andwere developed in ThermoScientific ECL reagent (Catalog #32106) for 1minute. Blots were analyzed using a ProteinSimple fluorescence imager,and images were saved and analyzed with Adobe Photoshop software.

FIG. 13 shows Western Blot analysis of culture supernatants fromrepresentative 293T transient transfections. Prior to transfection, 293Tcells were plated to 60 mm dishes at a density of 1.5×10e⁶ cells/plateand grown overnight. Transfection media was prepared according tomanufacturer's directions using Polyfect transfection reagent (QIAGEN,Valencia, Calif.). Positive and negative controls (THER4 and mocktransfection/no DNA, respectively) were included in each transfectionseries. Three different ApoA1-lnk-hIgG-PON1 plasmids encoding sequencevariants (THER4PON1 192Q, THER4PON1 192R, and THER4PON1 192K) weretransfected in addition to the THER4 plasmid and the mock transfection.Supernatant samples (10 μl) were loaded onto 4-12% Bis-Tris gels in 1×reducing LDS sample buffer in the order listed from left to right: mocktransfection, THER4, THER4PON1 192Q, THER4PON1 192R, THER4PON1 192K, andPrecision Plus Kaleidoscope molecular weight markers (BioRad, Hercules,Calif.). The lanes with the ApoA1-lnk-hIgG-PON1 fusion proteins containa single band migrating at approximately 105 kDa.

Stable transfection of ApoA1-lnk-hIgG-PON1 fusion protein expressionplasmids was performed by electroporation of a selectable, amplifiableplasmid, pDG, containing the constructs under the control of the CMVpromoter, into Chinese Hamster Ovary (CHO) CHO DG44 cells. Plasmid DNA(200 μg) was prepared using QIAGEN HISPEED® maxiprep kits, and purifiedplasmid was linearized at a unique Ascl site (New England Biolabs,Ipswich, Mass. Catalog #R0558), purified by phenol extraction(Sigma-Aldrich, St. Louis, Mo.), ethanol precipitated, washed, andresuspended in EX-CELLO 302 tissue culture media, (Catalog #14324,SAFC/Sigma Aldrich, St. Louis, Mo.). Salmon sperm DNA (Sigma-Aldrich,St. Louis, Mo.) was added as carrier DNA just prior to phenol extractionand ethanol precipitation. Plasmid and carrier DNA were coprecipitated,and the 400 μg was used to transfect 2×10⁷ CHO DG44 cells byelectroporation.

For transfection, CHO DG44 cells were grown to logarithmic phase inEX-CELL 302 complete media (see Example 4). Media for untransfectedcells and cells to be transfected also contained HT (diluted from a 100×solution of hypoxanthine and thymidine) (Invitrogen/Life Technologies,Grand Island, N.Y.). Electroporations were performed at 280 volts, 950microFarads, using a BioRad (Hercules, Calif.) GENEPULSER®electroporation unit with capacitance extender. Electroporation wasperformed in 0.4 cm gap sterile, disposable cuvettes. Electroporatedcells were incubated for 5 minutes after electroporation prior totransfer of culture to non-selective EX-CELL 302 complete media in T75flasks.

Transfected cells were allowed to recover overnight in non-selectivemedia prior to selective plating in 96 well flat bottom plates (Costar)at varying serial dilutions ranging from 250 cells/well (2500 cells/ml)to 2000 cells/well (20,000 cells/ml). Culture media for cell cloning wasEX-CELL 302 complete media containing 50 nM methotrexate. Transfectionplates were fed at five day intervals with 80 μl fresh media. After thefirst couple of feedings, media was removed and replaced with freshmedia. Plates were monitored and individual wells with clones were feduntil clonal outgrowth became close to confluent, after which cloneswere expanded into 24 well dishes containing 1 ml media. Aliquots of theculture supernatants from the original 96 well plate were also harvestedand frozen for later ELISA analysis to estimate IgG concentrations.

Screening Culture Supernatants for Production Levels of RecombinantFusion Proteins

Once clonal outgrowth of initial transfectants was sufficient, serialdilutions of culture supernatants from master wells were thawed and thedilutions screened for expression of Ig fusion protein by use of an IgGsandwich ELISA. Briefly, NUNC MAXISORP® plates were coated overnight at4° C. with 2 μg/ml F(ab′2) goat anti-human IgG (Jackson Immunoresearch,West Grove, Pa.; Catalog #109-006-098) in PBS. Plates were blocked inPBS/3% BSA, and serial dilutions of culture supernatants incubated atroom temperature for 2-3 hours or overnight at 4° C. Plates were washedthree times in PBS/0.05% Tween 20, and incubated with horseradishperoxidase conjugated F(ab′2)goat anti-human IgG (JacksonImmunoresearch, West Grove, Pa., Catalog #109-036-098) at1:7500-1:10,000 in PBS/0.5% BSA, for 1-2 hours at room temperature.Plates were washed five times in PBS/0.05% Tween 20, and bindingdetected with SUREBLUE RESERVE™ TMB substrate (SeraCare, Gaithersburg,Md.). Reactions were stopped by addition of equal volume of 1N HCl, andabsorbance per well on each plate was read at 450 nm on a SYNERGY™ HTplate reader (Biotek, Winooski, Vt.). Concentrations were estimated bycomparing the OD450 of the dilutions of culture supernatants to astandard curve generated using serial dilutions of a known standard, aprotein A purified human IgG fusion protein with an Ig tail identical tothat of the clones. Data was collected and analyzed using GENS™ software(Biotek, Winooski, Vt.) and Microsoft Office EXCEL® (Microsoft, Redmond,Wash.) spreadsheet software and GraphPad Prism 4.0 (GraphPad Software,La Jolla, Calif.).

For the THER4PON1 variants, 75-100 clones from each transfection werescreened for expression level. The expression level of the best clonesranged from 7.5-35 μg/ml in the initial 96 well culture supernatants.Clones with the highest level of expression were expanded up inExce11302 selective media and cell samples (approximately 1-2×10⁶ cells)frozen in liquid nitrogen. In addition, cultures were grown for ten daysand culture supernatants from spent cultures were harvested bycentrifugation, culture media filtered, and pH adjusted with sodiumcarbonate/bicarbonate solution to pH 8.0. Proteins from transfected CHOcells were purified by protein A affinity chromatography, using amodified purification strategy to preserve protein function. A modifiedstrategy was necessary for affinity chromatography of the THER4PON1fusion proteins, since both the apo A-1 and the PON1 specificities aresensitive to pH and to divalent cation conditions. Culture supernatantswere incubated in sterile 50 ml conical tubes with approximately 1.0 mlprotein A slurry (Repligen, Waltham, Mass.), per tube, with gentlerotation at 4° C., for 24-48 hours. Protein A slurry was harvested fromeach tube by centrifugation at 2400 rpm for 10 minutes, and culturemedia removed to a separate bottle. Slurry was sometimes incubated witha second batch of culture supernatant for another 24-48 hours, withgentle rotation at 4° C. Tubes were then centrifuged at 2400 rpm, 10minutes at 4° C. prior to removal of culture supernatants to secondarycontainer. The protein A resin was gently mixed in remaining culturesupernatant and transferred and loaded into presterilized, acid washedeconocolums (BioRAD, Hercules, Calif.) fitted with a two way stopcock.Columns were then washed in several column volumes Pierce/ThermoFisher(Rockville, Ill.) gentle antigen/antibody binding buffer, pH 8.0,allowing the buffer to drip through columns by gravity flow. Boundfusion proteins eluted with gentle antigen/antibody elution buffer pH6.6, (Pierce/ThermoFisher, Rockville Ill.). Eluted fractions (1.0ml/tube) were collected into microfuge tubes and aliquots from eachfraction assessed for protein level using a Nanodrop 2.0 (ThermoFisher,Waltham, Mass.) spectrophotometer. Positive fractions were pooled anddialyzed in FLOAT-A-LYZER® dialysis units with a molecular weight cutoffof 10 kilodaltons (Spectrum Labs/Repligen, Rancho Dominguez, Calif.).Dialysis buffer contained 0.9% NaCl, 2.5 mM HEPES buffer, 1 mM CaCl₂,and 5 mM sodium bicarbonate, pH 7.5. Dialysis was performed at 4° C., insterile roller bottles containing 2 liters buffer. A second round ofdialysis was performed in the same volume buffer. Proteins wereharvested after dialysis, concentrated using Millipore Sigma(Burlington, Mass.) Amicon centrifugal concentrators, and sterilefiltered through 0.2 μm PES syringe filter units. Protein concentrationswere then determined from the OD280 assayed using a Nanodropspectrophotometer with dialysis buffer as a blank. Once THER4PON1 fusionmolecules were purified, they were assessed by SDS-PAGE analysis,Western blotting, and in further functional assays.

Example 11: Measurement of PON1 Enzyme Activity in THER4PON1 192Sequence Variants

PON1 has multiple enzyme activities. Here, enzyme activity was screenedby two different assays; a fluorescent kinetic assay which measuresphosphotriesterase activity associated with organophosphate pesticides,while the second assay assesses the arylesterase activity of the enzyme.

Phosphotriesterase Activity

FIG. 14 shows the results of a PON1 functional assay for thephosphotriesterase activity using culture supernatants from transfectedclones of three THER4PON1 sequence variants as test enzymes: THER4PON1(also referred to herein as “THER4PON1 192Q” or “T4P1-192Q”), THER4PON1Q192R (also referred to herein as “THER4PON1 192R” or “T4P1-192R”), andTHER4PON1 Q192K (also referred to herein as “THER4PON1 192K” or“T4P1-192K”) (see Examples 8 and 10). The data were generated using acommercially available kit from Molecular Probes/Life Technologies, theEnzChek paraoxonase assay kit E33702, (ThermoFisher). This kit providesa sensitive, less toxic, homogeneous fluorometric assay(excitation/emission maxima ˜360/450 nm) for the organophosphataseactivity of paraoxonase and is based on the hydrolysis of a proprietary,fluorogenic organophosphate analog as substrate. Enzyme assays were setup with the stock solutions provided, and with serial dilutions of apositive control PON1 enzyme provided with the kit reagents, or withserial dilutions of dialyzed, filtered CHO culture supernatants of thethree different sequence variants of ApoA1-lnk-hIgG-PON1 fusion proteinsas test enzymes. The relative fluorescence units produced as a functionof time were determined for each reaction using a fixed concentration ofsubstrate and serial dilutions of the different enzyme supernatants.Enzyme kinetics were measured using a SYNERGY™ HT fluorescence platereader (BioTek, Winooski, Vt.), and the data was analyzed using GENS™software. The data shown demonstrate that the T4P1 192 sequence variantsall exhibit organophosphatase activity detectable in unconcentrated,filtered cell culture supernatants after transfection.

Each THER4PON1 192 sequence variant was purified from spent CHO culturesupernatants using a modified protein A purification strategy asdescribed previously. The EnzCheck paraoxonase assay was then repeatedwith purified protein to better assess the specific activity of thefusion proteins. Serial dilutions of the purified T4P1-192Q, T4P1-192R,T4P1-192K, and THER4 were prepared and transferred to appropriate wellsof 96 well, black microplates. Similar dilution series were alsoprepared for the fluorescence reference standard and theorganophosphatase positive control enzyme provided with the kit.Organophosphatase substrate was prepared according to the kitinstructions and aliquots added to each well of the assay except for thefluorescence reference wells. For these assays, the concentration oforganophosphate substrate was kept fixed for every reaction. Afteraddition of substrate, plates were transferred to a SYNERGY™ HT platereader at 37° C., with excitation 360 nm and emission 460 nm settings.Serial dilutions of the control and test enzymes were assessed for theRFU generated during a 60 minute kinetic assay, with readings every 60seconds (1 min). The RFU as a function of time was assessed relative tothe fluorescent reference standard readings and to serial dilutions ofthe organophosphatase positive control supplied with the kit. Inclusionof the fluorescence reference standard permitted proper setting of thereader gains and sensitivity settings, and generation of a standardcurve for estimation of activity per unit volumes. The graph shown inFIG. 15 displays relative fluorescence units plotted as a function oftime (HH:MM:SS), comparing the activity profiles of the controlorganophosphatase enzyme at two different dilutions (50 mU and 20 mU) tothat of the T4P1-192K, T4P1-192R, and T4P1-192Q molecules at 415 nM, orto that of the THER4 molecule at 715 nM. The equation of the line fit tothe stand curve was determined and used to convert the fluorescencemeasures to the nmol of fluorescent product. The 30 minute time pointwas used for these calculations. The U/L estimates of the activity forthe test proteins and the positive controls were 1688 U/L for T4P1-192Q,3943 U/L for T4P1-192R, 10840 U/L for T4P1-192K, and 1600-11900 U/L forthe organophosphatase positive control dilution series from 10mU to100mU.

Arylesterase Activity

In addition to assessment of the paraoxonase fusion proteins fororganophosphatase activity, the arylesterase activity was also measuredusing conversion of phenyl acetate (Sigma-Aldrich) to phenol. Purifiedfusion proteins were diluted in enzyme assay buffer, 20 mM Tris-HCl (pH7.5), 2 mM CaCl₂ to 80 μg/ml (2× enzyme for each dilution). Two-foldserial dilutions were then made of the T4P1-192Q, T4P1-192R, andT4P1-192K variants and of the THER4 fusion protein, using the samebuffer to create a dilution series of each molecule. The serialdilutions of enzyme were used in assays with a constant amount of 5 mMphenyl acetate as substrate (10 mM stock, diluted 2×) to determine theoptimal amount of enzyme to be used with serial dilutions of substrate.A kinetic enzyme assay was performed by aliquoting 50 μl/well eachserial dilution of enzyme into a 96 well UV transparent plate (GreinerBioOne, Catalog #655 801), followed by addition of 50 μl 2× substratesolution containing 10 mM phenyl acetate, to give a final concentrationof 5 mM. The conversion of substrate was monitored by measuring the ODat 270 nm at 60 second intervals for 20 minutes. For each well, akinetic curve was generated reflecting the rate of substrate conversion.FIG. 16 shows representative results of the arylesterase analysis usingthe THER4PON1 192Q, 192R, and 192K molecules and the THER4 negativecontrol as test enzymes for conversion of the phenyl acetate substrateto phenol and acetic acid. T4P1-192Q and T4P1-192R have similar kineticsunder these conditions of low salt, although the 192Q form converted thesubstrate more efficiently than the 192R variant. The T4P1-192K variantat the same concentration (208 nM) exhibits a much higher enzymeefficiency than either of the 192Q and 192R forms. At lowerconcentrations (104 nM), this variant looked more similar to the othertwo forms, but still showed the highest activity. The THER4 fusionprotein without PON1 did not generate a signal for conversion of thephenyl acetate substrate in this assay. For each T4P1 variant, theenzyme concentration which generated a kinetics curve most closelyapproximating linearity was used as the enzyme concentration in a secondassay, keeping enzyme concentration fixed while varying the amount ofsubstrate. Serial dilutions of phenyl acetate substrate were then madefrom 0.5 mM to 20 mM with a fixed concentration of fusion protein asenzyme in order to further characterize the enzyme kinetics for thesemolecules.

Example 12: In Vivo Analysis of the Pharmacokinetics of THER4PON1 192Rin Wild-Type Mice

A PK study was performed on the THER4PON1 192R protein to determine therate of clearance of the molecule in vivo. This study was performed inwild-type C57BL/6 mice using intravenous (IV) injection of the testmaterials. THER4PON1 192R protein (see Examples 8 and 10) was purifiedby a modified protein A purification strategy, followed by testing ofthe purified proteins prior to the in vivo study.

C57BL/6 mice (5 mice per group) were injected intravenously with 200μg/mouse fusion protein in a volume of 0.1 ml or with vehicle in asingle bolus injection. Mice were then sacrificed at 1 hour, 6 hours, 24hours, 72 hours, 7 days, and 14 days after injection. Plasma washarvested from each mouse at these time points and assessed by sandwichELISA for levels of the human fusion protein. Trial ELISAs wereperformed with wild-type mouse sera spiked with human fusion protein inorder to determine the parameters for testing the treated samples.Fusion protein levels were screened by a sandwich ELISA using anti-humanIgG to capture the molecules (2.0 μg/ml in D-PBS) from mouse plasma, andusing either an HRP-conjugated anti-human IgG (Jackson Immunoresearch,Catalog #109-036-098 at 1:7500) or HRP-conjugated anti-human APO A-1(ThermoFisher Scientific, Catalog #PA1-28965 at 1:1500) antibody fordetection. ELISA plates were washed with 1×D-PBS containing 0.05%Tween-20 between each step. SeraCare (Milford, Mass.; Catalog#5120-0076) SUREBLUE RESERVE™ TMB 1-component microwell peroxidasesubstrate (85 μl/well) was used for visualization, and reactions stoppedby the addition of 1N HCl. ELISA plates were analyzed on a SYNERGY™ HTplate reader at wavelengths of 450 and 630 nm. Raw data was importedinto GraphPad Prism 4 for analysis.

FIG. 17 shows results of the PK analysis of the purifiedApoA1-lnk-hIgG-PON1 fusion protein after injection into wild-type mice.The data shown summarize the plasma levels of the THER4PON1 192R variantin wild-type mice at the indicated time points after injection. Alsoshown is the absence of signal from five mice injected with vehiclealone. The dot plot graph summarizes the concentration estimates foreach individual mouse. Each dot represents the estimate for a singlemouse. These concentration estimates were generated from the ELISA datausing serial dilutions and replicates of each plasma sample compared toa standard curve created from serial dilutions of the purified,uninjected treatment molecule. Five animals were assessed for presenceof the T4P1-192R purified protein in plasma per time point. The datashow that low levels of the fusion protein are still detectable in mouseplasma after 14 days. The dot plot displays the concentration data as afunction of time using a linear scale. A similar dot plot can begenerated that displays the same data using a logarithmic scale for theconcentration estimates. In this case, the dot plot generates a curvethat more closely approximates linearity, but still departs from it. Thepharmacokinetics exhibit second order or higher behavior, suggesting arapid initial loss due to localization in the periphery, followed by amore gradual decrease due to metabolism. This more complex curvesuggests a two compartment or higher process of elimination. TheTHER4PON1 variants are expected to interact with HDL, FcR, and possiblyother apolipoprotein targets in addition to the substrate targets forPON1, so that much of the initial bolus may localize to sites in theperiphery. Other systemic reservoirs including monocytes may bind to theTHER4PON1 192R variant, resulting in a rapid decrease in circulatingplasma levels during the first few hours after injection.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

What is claimed is:
 1. A polynucleotide encoding a fusion polypeptide,wherein the fusion polypeptide comprises, from an amino-terminalposition to a carboxyl-terminal position, ApoA1-L1-D-L2-P, wherein:ApoA1 is a first polypeptide segment comprising an amino acid sequencehaving at least 95% identity with amino acid residues 19-267 or 25-267of SEQ ID NO:2, wherein said first polypeptide segment has cholesterolefflux activity; L1 is a first polypeptide linker comprising at least 5amino acid residues; D is an immunoglobulin Fc region; L2 is a secondpolypeptide linker; and P is a paraoxonase having at least 98% identitywith amino acid residues 16-355 of SEQ ID NO:12, amino acid residues16-355 of SEQ ID NO:42, or amino acid residues 16-355 of SEQ ID NO:44.2. The polynucleotide of claim 1, wherein the first polypeptide segmenthas the amino acid sequence shown in residues 19-267 or 25-267 of SEQ IDNO:2.
 3. The polynucleotide of claim 1, wherein L1 comprises at least 16amino acid residues.
 4. The polynucleotide of claim 1, wherein L1consists of from 10 to 60 amino acid residues.
 5. The polynucleotide ofclaim 1, wherein L1 comprises two or more tandem repeats of the aminoacid sequence of SEQ ID NO:15.
 6. The polynucleotide of claim 1, whereinthe Fc region is a human Fc region.
 7. The polynucleotide of claim 6,wherein the human Fc region is a γ1 Fc variant comprising one or moreamino acid substitutions relative to a wild-type human γ1 Fc regionsequence.
 8. The polynucleotide of claim 7, wherein the Fc region is ahuman γ1 Fc variant in which residues C220, C226, and C229, according toEU numbering for human IgG heavy chain constant region, are eachreplaced by serine.
 9. The polynucleotide of claim 8, wherein residueP238, according to EU numbering for human IgG heavy chain constantregion, is replaced by serine.
 10. The polynucleotide of claim 9,wherein residue P331, according to EU numbering for human IgG heavychain constant region, is replaced by serine.
 11. The polynucleotide ofclaim 1, wherein L2 has the amino acid sequence shown in residues526-543 of SEQ ID NO:28.
 12. The polynucleotide of claim 1, wherein theparaoxonase has the amino acid sequence shown in residues 16-355 of SEQID NO:12, residues 16-355 of SEQ ID NO:42, or residues 16-355 of SEQ IDNO:44.
 13. The polynucleotide of claim 1, wherein the encoded fusionpolypeptide comprises an amino acid sequence having at least 99%identity with (i) residues 19-883 or 25-883 of SEQ ID NO:28, (ii)residues 19-873 or 25-873 of SEQ ID NO:38, (iii) residues 19-883 or25-883 of SEQ ID NO:46, or (iv) residues 19-883 or 25-883 of SEQ IDNO:48.
 14. The polynucleotide of claim 13, wherein the encoded fusionpolypeptide comprises the amino acid sequence shown in (i) residues19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46, or (iv)residues 19-883 or 25-883 of SEQ ID NO:48.
 15. A method of making afusion polypeptide, the method comprising: (a) culturing a mammaliancell into which has been introduced an expression vector comprising thefollowing operably linked elements: (i) a transcription promoter; (ii) aDNA segment encoding a fusion polypeptide, wherein the fusionpolypeptide comprises, from an amino-terminal position to acarboxyl-terminal position, ApoA1-L1-D-L2-P, wherein: ApoA1 is a firstpolypeptide segment comprising an amino acid sequence having at least95% identity with amino acid residues 19-267 or 25-267 of SEQ ID NO:2,wherein said first polypeptide segment has cholesterol efflux activity;L1 is a first polypeptide linker comprising at least 5 amino acidresidues; D is an immunoglobulin Fc region; L2 is a second polypeptidelinker; and P is a paraoxonase having at least 98% identity with aminoacid residues 16-355 of SEQ ID NO:12, amino acid residues 16-355 of SEQID NO:42, or amino acid residues 16-355 of SEQ ID NO:44; and (iii) atranscription terminator, wherein the cell expresses the DNA segment andthe encoded fusion polypeptide is produced; and (b) recovering thefusion polypeptide.
 16. The method of claim 15, wherein the human Fcregion is a γ1 Fc variant comprising one or more amino acidsubstitutions relative to a wild-type human γ1 Fc region sequence. 17.The method of claim 15, wherein the paraoxonase has the amino acidsequence shown in residues 16-355 of SEQ ID NO:12, residues 16-355 ofSEQ ID NO:42, or residues 16-355 of SEQ ID NO:44.
 18. The method ofclaim 15, wherein the encoded fusion polypeptide comprises an amino acidsequence having at least 99% identity with (i) residues 19-883 or 25-883of SEQ ID NO:28, (ii) residues 19-873 or 25-873 of SEQ ID NO:38, (iii)residues 19-883 or 25-883 of SEQ ID NO:46, or (iv) residues 19-883 or25-883 of SEQ ID NO:48.
 19. The method of claim 18, wherein the encodedfusion polypeptide comprises the amino acid sequence shown in (i)residues 19-883 or 25-883 of SEQ ID NO:28, (ii) residues 19-873 or25-873 of SEQ ID NO:38, (iii) residues 19-883 or 25-883 of SEQ ID NO:46,or (iv) residues 19-883 or 25-883 of SEQ ID NO:48.
 20. The method ofclaim 15, wherein the encoded fusion polypeptide is produced in the celland recovered as a dimeric protein.